Methods of determining cellular chemosensitivity

ABSTRACT

The present invention provides methods of determining cell sensitivity to a therapeutic agent.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 13/478,831, filedMay 23, 2012, which is a continuation of U.S. Ser. No. 11/695,321, filedApr. 2, 2007, which claims the benefit U.S. Ser. No. 60/788,138, filedMar. 31, 2006, the contents of each of which are incorporated herein byreference in their entirety.

FIELD OF THE INVENTION

The invention relates to generally methods of determining cellularchemosensitivity by determining the pattern of sensitivity of a cell toa panel of BH3 domain peptides.

BACKGROUND OF THE INVENTION

Programmed cell death, referred to as apoptosis, plays an indispensablerole in the development and maintenance of tissue homeostasis within allmulticellular organisms (Raff, Nature 356: 397-400, 1992). Genetic andmolecular analysis from nematodes to humans has indicated that theapoptotic pathway of cellular suicide is highly conserved (Hengartnerand Horvitz, Cell 76: 1107-1114, 1994). In addition to being essentialfor normal development and maintenance, apoptosis is important in thedefense against viral infection and in preventing the emergence ofcancer.

Diverse intrinsic death signals emanating from multiple subcellularlocales all induce the release of cytochrome c from mitochondria toactivate Apaf-1 and result in effector caspase activation. Proteins inthe BCL-2 family are major regulators of the commitment to programmedcell death as well as executioners of death signals at themitochondrion. Members of this family include both pro- andanti-apoptotic proteins and share homology in up to four conservedregions termed BCL-2 homology (BH) 1-4 domains (Adams and Cory, 1998).The family can be divided into three main sub-classes. Theanti-apoptotic proteins, which include BCL-2 and BCL-X_(L), are all“multidomain,” sharing homology throughout all four BH domains. However,the pro-apoptotic proteins can be further subdivided and includemultidomain proteins, such as BAX and BAK, which possess sequencehomology in BH1-3 domains. The more distantly related “BH3-only”proteins are to date all pro-apoptotic and share sequence homologywithin the amphipathic α-helical BH3 region, which is required for theirapoptotic function (Chittenden et al., 1995; O'Connor et al., 1998; Wanget al., 1996; Zha et al., 1997).

Multidomain pro-apoptotic proteins such as BAX and BAK upon receipt ofdeath signals participate in executing mitochondrial dysfunction. Inviable cells, these proteins exist as monomers. In response to a varietyof death stimuli, however, inactive BAX, which is located in the cytosolor loosely attached to membranes, inserts deeply into the outermitochondrial membrane as a homo-oligomerized multimer (Eskes et al.,2000; Gross et al., 1998; Wolter et al., 1997). Inactive BAK resides atthe mitochondrion where it also undergoes an allosteric conformationalchange in response to death signals, which includes homo-oligomerization(Griffiths et al., 1999; Wei et al., 2000). Cells deficient in both BAXand BAK are resistant to a wide variety of death stimuli that emanatefrom multiple locations within the cell (Wei et al., 10 2001).

The BH3-only molecules constitute the third subset of this family andinclude BID, NOXA, PUMA, BIK, BIM and BAD (Kelekar and Thompson, 1998).These proteins share sequence homology only in the amphipathic a-helicalBH3 region which mutation analysis indicated is required inpro-apoptotic members for their death activity. Moreover, the BH3-onlyproteins require this domain to demonstrate binding to “multidomain”BCL-2 family members. Multiple binding assays, including yeasttwo-hybrid, co-immunoprecipitation from detergent solubilized celllysates and in-vitro pull down experiments indicate that individualBH3-only molecules display some selectivity for multidomain BCL-2members (Boyd et al., 1995; O'Connor et al., 1998; Oda et al., 2000;Wang et al., 1996; Yang et al., 1995). The BID protein bindspro-apoptotic BAX and BAK as well as anti-apoptotic BCL-2 and BCL-X_(L)(Wang et al., 1996; Wei et al., 2000). In contrast, BAD, and NOXA asintact molecules display preferential binding to anti-apoptotic members(Boyd et al., 1995; O'Connor et al., 1998; Oda et al., 2000; Yang etal., 1995)

SUMMARY OF THE INVENTION

In various aspects, the invention provides methods of predictingsensitivity of a cell to a therapeutic agent by contacting the cell or acellular component (e.g., mitochondria) thereof with a BH3 domainpeptide and detecting apoptosis. The presence of apoptosis indicatesthat the cell is sensitive to the therapeutic agent. Alternatively,sensitivity of a cell to a therapeutic agent is determined by providinga BH3 profile of the cancer cell and comparing the BH3 profile to a 30control profile. A similarity of the BH3 profile in the cancer cellcompared to the control profile indicates the cancer cell is sensitiveto the therapeutic agent.

Also provided are methods of selecting an agent that is therapeutic fora subject by providing a cancer cell or cellular component thereof,contacting the cell or cellular component with a BH3 domain peptide ormimetic thereof and determining whether or not the BH3 domain peptide ormimetic induces apoptosis in the cancer cell or cellular componentthereof to produce a test BH3 profile. The test BH3 profile is comparedwith a therapeutic agent BH3 profile. A similarity of the test BH3profile compared to the therapeutic agent BH3 profile indicates that theagent is therapeutic for the subject.

Apoptosis is detected for example by detecting cytochrome c release frommitochondria. The therapeutic agent is a chemotherapeutic agent a BH3domain mimetic, or antagonist of an anti-apoptotic protein. The BH3domain peptide is derived from the BH3 domain of a BID, a BIM, a BAD, aBIK, a NOXA, a PUMA a BMF, or a HRK polypeptide. Exemplary BH3 domainpeptides include SEQ ID NO: 1-14 and 15. The BH3 domain peptide is anactivator or a sensitizer of apoptosis. Preferably, the BH3 domainpeptide is a sensitizer.

A profile containing a pattern of mitochondrial sensitivity to BH3peptides taken from one or more subjects who have cancer is alsoprovided by the invention.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting. Other features and advantages of the invention will beapparent from the following detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a bar chart showing the effects of sensitizer BH3 peptides oncytochrome c release from mouse liver mitochondria. Peptideconcentrations were 10 uM unless otherwise indicated; tBID concentrationwas 13 nM. Average and standard deviation from at least threeindependent assays performed for each antiapoptotic protein are shown.

FIG. 1B is a bar chart showing the effects of tBID (first bar) and BCL-2(1.2 uM, second bar) on cytochrome c release from mitochondria. Theeffect of sensitizer BH3 peptides on restoration of cytochrome c releaseis also shown. Note that in each case, restoration of cytochrome crelease corresponds to a high affinity interaction in Table 2B.

FIG. 1C is a bar chart showing the effects of tBID (first bar) andBCL-XL (0.25 uM, second bar) on cytochrome c release from mitochondria.The effect of sensitizer BH3 peptides on restoration of cytochrome crelease is also shown.

FIG. 1D is a bar chart showing the effects of tBID (first bar; note tBIDconcentration was 43 nM in this experiment) and BCL-w (6.3 uM; secondbar) on cytochrome c release from mitochondria. The effect of sensitizerBH3 peptides on restoration of cytochrome c release is also shown.

FIG. 1E is a bar chart showing the effects of tBID (first bar) and MCL-1(1.1 uM; second bar) on cytochrome c release from mitochondria. Theeffect of sensitizer BH3 peptides on restoration of cytochrome c releaseis also shown.

FIG. 1F is a bar chart showing the effects of tBID (first bar) and BFL-1(2.4 uM; second bar) on cytochrome c release from mitochondria. Theeffect of sensitizer BH3 peptides on restoration of cytochrome c releaseis also shown.

FIG. 2 is a bar chart showing the effect of BH3 peptides on cytochrome crelease. MLM were treated with the indicated peptides at 10 uM in thepresence or absence of 0.2 uM BCL-XL protein.

FIG. 3A is a photograph of a Western blot showing the results of aGST-pulldown assay in which GST-BCL-w (or point mutant R96P) wascombined with tBID protein and the indicated BH3 peptides (10 μM). TheBH1 domain R96P mutant of BCL-w lacks the ability to bind BH3 domains.For convenience, BCL-w binding pattern from Table 2B is excerpted below.

FIG. 3B and FIG. 3C are line graphs showing the effects of BAD and NOXABH3 peptides on displacement of a fluorescein tagged BIM BH3 peptidefrom BCL-2 and MCL-1 proteins by fluorescence depolarization. Shown arerepresentative plots from three independent experiments for eachcombination.

FIG. 4A is a line graph showing the effects of IL-3 withdrawal on thesurvival of wtFL5.12 and FL5.12-BCL-2 cells. Survival was imputed forcells not staining with Annex in V by FACS analysis. Shown is averageand standard deviation of three independent experiments.

FIG. 4B is a line graph showing the effects of ABT-737, a BCL-2antagonist, on the survival of IL-3 replete and IL-3 starvedFL5.12-BCL-2 cells. Viability was assayed by absence of Annex in Vstaining. Shown is average and standard deviation of three independentexperiments.

FIG. 4C is a bar chart showing the effects of ABT-737 and ZVAD.fmk onthe survival of FL5.12 cells (top). Also, a photograph of an immunoblotshows the effect of ABT-737 on PARP cleavage (bottom).

FIG. 5 is a bar chart showing the effect of ABT-737 and ZVAD.fmk onFL5.12 viability. FL5.12 were grown either with IL-3 or in the absenceof IL-3 for 24 hours, then treated as indicated for either 30 minutes or1, 2, or 3 hours. Cell death was measured by Annexin V staining via FACSanalysis.

FIG. 6A is a bar chart showing the effects of BH3 peptides (10 uM) oncytochrome c release from mitochondria isolated from wtFL5.12 cellsgrown in the presence of IL-3 (no fill bars); FL5.12-BCL-2 cells grownin the presence (speckled bars) or absence (slashed bars) of IL-3 for 24hours. Shown is average and standard deviation of three independentexperiments. For convenience, BCL-2 binding pattern from Table 2B isexcerpted below.

FIG. 6B is a bar chart showing the effects of NOXA and BAD BH3 oncytochrome c release from mitochondria isolated from wt and BCL-2 FL5.12cells grown in the presence of IL-3. Shown is average and standarddeviation of three independent experiments. FIG. 6C is a photograph of aWestern blot depicting the effects of NOXA A, BAD, ABT-737, or controlenantiomer on cytochrome c release from FL5.12 cells grown in theabsence of IL-3 for 24 hours.

FIG. 6D shows photographs of immunoblots depicting the effects of IL-3withdrawal on BIM levels in FL512-BCL-2 whole cell lysates (left) andsamples immunoprecipitated by an antibody directed against the humanBCL-2 transgene product (right). Numbers at top refer to hours afterIL-3 withdrawal. Control lane performed without antihuman BCL-2 antibodyin pulldown at right.

FIG. 6E is a photograph of a Western blot showing the results of animmunoprecipitation assay. BAX was immunoprecipitated using an antibodyrecognizing all BAX conformations (Δ21) or only the activatedconformation with N-terminal exposure (NT). Death induced in the cellsis indicated below. At right, mitochondria isolated from IL-3 starvedcells were treated with ABT-737 or control enantiomer, andimmunoprecipitation with NT performed as indicated. CD56 indicatescontrol immunoprecipitation by an irrelevant antibody recognizing CD56.

FIG. 7A is a line graph showing the effects of MCL-1 and BCL-2 on celldeath induced by dexamethasone in 2B4 cells. Viability determined byabsence of Annexin V staining by FACS. Shown is average and standarddeviation of three independent experiments.

FIG. 7B is a line graph showing the effects of BCL-2 antagonist ABT-737on MCL-1 dependent 2B4 cells and BCL-2 dependent 2B4 cells. Shown isaverage and standard deviation of three independent experiments.

FIG. 7C is a bar chart showing the effects of BH3 peptides on cytochromec release from mitochondria isolated from MCL-1-expressing 2B4 cellstreated as indicated. Shown is average and standard deviation of threeindependent experiments. For convenience, MCL-1 binding pattern fromTable 2B is excerpted below.

FIG. 7D is a bar chart showing the effects of BH3 peptides on cytochromec release from mitochondria isolated from BCL-2-expressing 2B4 cellstreated as indicated. For convenience, BCL-2 binding pattern form Table2B is excerpted below.

FIG. 7E is a photograph of an immunoblot showing the effects ofdexamethasone on FLAG-MCL-1 transfected 2B4 cells. FLAG antibody linkedto agarose beads immunoprecipitated proteins complexing with FLAG-MCL-1.Increased BIM sequestration by MCL-1 correlates with MCL-1 dependence.

FIG. 7F is a photograph of an immunoblot showing the effects ofdexamethasone on FLAG-BCL-2 transfected 2B4 cells.

FIG. 7G is a bar chart showing the effects of BH3 peptides ondexamethasone-treated FLAG-MCL-1 2B4 cells. Primed FLAG-MCL-1 2B4 cellstransfected with BH3 peptides illustrate an MCL-1 pattern.

FIG. 8A is a bar chart showing the effects of BH3 peptides (10 μM,unless otherwise noted) on cytochrome c release from mitochondriaisolated from liver.

FIG. 8B is a bar chart showing the effects of BH3 peptides on cytochromec release from mitochondria isolated from BCL-2-dependent leukemia. Forconvenience, BCL-2 binding pattern from Table 2B is excerpted below.Shown is average and standard deviation of three independentexperiments, except 30 and 100 μM treatments which were performed once.

FIG. 8C is a picture of an immunoblot of samples from a BCL-2 dependentleukemia. First lane shows a whole cell lysate, 25 ug loaded; secondlane—products of an immunoprecipitation using an antibody against thehuman BCL-2 transgene product; third lane—a control with Protein A beadsalone; fourth lane—control immunoprecipitation using an irrelevanthamster monoclonal antibody recognizing murine CD-40.

FIG. 8D is a bar chart showing the effects of BH3 peptides on cytochromec release from mitochondria isolated from two SCLC cell lines, H146 andH1963. N=3 for each and the error bars represent the standard deviation.

FIG. 9 is a model of selective cancer sensitivity to sensitizer BH3mimetic treatment. Living unprimed cells (I) are primed for deathfollowing death stimuli (II). The leukemia cell is tonically primed fordeath without external intervention (II). Cells in the primed stateundergo apoptosis in response to antiapoptotic antagonists (III)—thosein the unprimed state do not. A) FL%.12-BCL-2. B) 2B4-MCL-1. C)myc/BCL-2 leukemia cells.

FIG. 10 is a diagram depicting a model of BCL-2 family control ofprogrammed cell death. Death signals cause induction orpost-translational activation of BH3-only proteins. Activator BH3-onlyproteins, including BID and BIM, induce oligomerization of BAX and/orBAK, causing MOMP, cytochrome c release and caspase activation resultingin cell death. Antiapoptotic proteins prevent apoptosis by sequesteringactivator BH3-only proteins and BAX/BAK, upstream of BAX/BAKoligomerization. Sensitizer BH3-only proteins promote cell death bybinding the antiapoptotic proteins, displacing activator BH3-onlyproteins to trigger BAX/BAK oligomerization.

FIG. 11 is a diagram depicting the intrinsic or mitochondrial programmedcell death pathway. In response to death signaling, activator BH3-onlyproteins are triggered to interact with BAX and BAK, inducing BAX andBAK oligomerization. This oligomerization is followed bypermeabilization of the mitochondrial outer membrane, which releasesproapoptotic factors like cytochrome c to the cytosol. Cytosoliccytochrome c forms a complex with APAF-1 and Caspase-9 to make theholoenzyme known as the apoptosome, which in turn activates effectorCaspase-3, leading to widespread proteolysis. This pathway can beinterrupted by antiapoptotic members like BCL-2, which can bindactivator BH3-only proteins, preventing their interaction with BAX andBAK. This inhibitory interaction can itself be antagonized by sensitizerBH3-only domains, which compete for the binding site in BCL-2,displacing activators bound by BCL-2.

FIG. 12A is a line graph showing the effect of ABT-737 on CLL cellviability. CLL cells from 24 patient samples were cultured for 48 hourswith different concentrations of compounds. Death was quantitated byAnnexin-V staining and normalized to solvent (DMSO) treated controls.

FIG. 12B is a line graph showing the effect of ABT-737 on CLL cellviability. CLL cells harvested from 5 patient samples were cultured for4 hours with different concentrations of compounds. Death wasquantitated as in FIG. 12A.

FIG. 12C is a line graph showing the effect of ABT-737 on normal PBMCviability. PBMC's were cultured for 24 hours in the presence of theindicated concentrations of compounds.

FIG. 13A is a photograph of an immunoblot showing BCL-2 and BIM proteinlevels in whole cell lysates from CLL samples (number corresponds topatient number in FIG. 12A). Three isoforms of BIM (BIM extralong-BIMEL, BIM long-BIML, and BIM short-BIMS) are shown.

FIG. 13B is a photograph of an immunoblot showing BCL-2 and BIM proteinlevels in whole cell lysates from PBMC. Three normal PBMC lysates are atleft, CLL lysates at right.

FIG. 13C is a photograph of an immunoblot showing BCL-2 and BIM proteinlevels in whole cell lysates from two independent CLL primary samplesmade at time of cell harvest (pre) and 48 hours post-culture (post).

FIG. 13D is a photograph of an immunoblot showing BCL-2 protein levelsin primary CLL cells (A-F) and primary follicular lymphoma cells (FL).The immunoblots are indexed to lysates from the t(14;18)-containing H2human lymphoma cell line.

FIG. 14 is a bar chart showing the effects of BH3-only domain peptides(100 μM) or compounds (100 μM) on cytochrome c release (measured byELISA) from mitochondria isolated from independent primary CLL patientsamples. BADmu=a point mutant of the BAD BH3-only domain, used as anegative control. N=7, except for BADmu where N=5, ABT-737 and negativecontrol enantiomer N=3. Error bars represent the standard deviation.

FIG. 15A is a photograph of an immunoblot of BCL-2 and BIM proteins inwhole cell lysates of seven independent CLL samples.

FIG. 15B is a photograph of an immunoblot of BCL-2 and BIM proteins inwhole cell lysates of primary CLL cells. Primary CLL cells were culturedfor 24 hours with 100 nM ABT-737, 100 nM negative control enantiomer, orvehicle (DMSO) +200 μM ZVAD.fmk. Death was then quantitated by Annexin-Vstaining. Immunoprecipitation (i.p.) using lysates from each treatmentgroup was performed using an anti-BCL-2 antibody. Results shown arerepresentative of three independent experiments.

FIG. 15C is a photograph of an immunoblot. A BCL-2 antibody was used toimmunoprecipitate BCL-2 from CLL lysates from four independent patientsamples. After rinsing detergent away, the complex bound to the beadswas then incubated with DMSO, 1 μM negative control enantiomer, or 1 μMABT-737. Shown is the resulting immunoblot of the fraction displaced tothe supernatant, probed for BIM.

FIG. 15D is a photograph of an immunoblot. Freshly isolated CLL cellswere incubated with DMSO, 10 nM, 100 nM, or 1 μM ABT-737 or negativecontrol enantiomer for 4 hours. % dead was assessed by Annexin-Vstaining. Oligomerization of BAX was evaluated by anti-BAX immunoblot ofchemically crosslinked whole cell lysates.

FIG. 15E is a bar chart showing the effect of 1% DMSO or 100 μM BAD BH3peptide on mitochondria isolated from CLL samples. Samples werepre-incubated with antibodies directed against either the human BIM BH3domain (Agent) or an irrelevant antigen (CD56) as indicated. N=5, barsshow+standard deviation.

FIG. 16A is a bar chart showing the effect of 100 μM BH3 peptides oncytochrome c release from mitochondria isolated from LP1 cells.

FIG. 16B is a photograph of an immunoblot of LP1 and L363 cell linescomparing levels of MCL-1, BCL-2, and BIM.

FIG. 16C is a line graph showing the effect of 48-hour treatment withABT-737 on the viability of L363 cells and LP1 cells. N=3, barsshow+standard deviation.

FIG. 17 is a diagram depicting a model of ABT-737 induced death at themitochondria. Mitochondrial BCL-2 sequesters BIM in CLL cells. Uponaddition of ABT-737, BIM is displaced and BCL-2 becomes occupied byABT-737. Freed BIM then interacts with BAX or BAK, inducingoligomerization leading to cytochrome c release and irreversiblecommitment to programmed cell death. BCL-2 primed with activatorBH3-only proteins renders the cancer cell sensitive to treatment withABT-737 and possibly other chemotherapeutic agents.

FIG. 18A is a chart showing the interaction pattern between BH3 peptidesand antiapoptotic proteins.

FIGS. 18B-18E are a series of bar charts showing BH3 profiles forvarious lymphoma cell lines.

FIGS. 19A-19B are a series of line graphs showing cell sensitivity tovarious agents

FIGS. 20A-20D are a series of bar charts showing a comparison ofmitochondrial and cell-based BH3 profiling.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based in part by the discovery of a thirdcellular state with respect to programmed cell death. This state hasbeen named “primed for death”. Until the present discovery, only twostates had been identified with respect to programmed cell death, aliveand dead. Cells that are primed for death require tonic antiapoptoticfunction for survival.

Using a panel of peptides derived from BH3 domains of BH3-only proteinsthat selectively antagonize individual BCL-2 family members BCL-2,BCL-XL, BCL-w, MCL-1 and BFL-1, it was shown that cellular “addiction”to individual antiapoptotic proteins can be diagnosed based onmitochondrial response to these peptides. This panel of peptides isshown in Table 1 and are referred to herein BH3 domain peptides.Antiapoptotic proteins BCL-2, BCLXL, MCL-1, BFL-1 and BCL-w each bear aunique pattern of interaction with this panel of proteins. Cellulardependence on an antiapoptotic protein for survival is decoded based onthe pattern of mitochondrial sensitivity to this peptide panel. Thisstrategy is called BH3 profiling.

Table 1 SEQ ID AMINO ACID SEQUENCE NO BID EDIIRNIARHLAQVGDSMDR  1 BIMMRPEIWIAQELRRIGDEFNA  2 BIDmut EDIIRNIARHAAQVGASMDR  3 BADLWAAQRYGRELRRMSDEFEGSFKGL  4 BIK MEGSDALALRLACIGDEMDV  5 NOXA AAELPPEFAAQLRKIGDKVYC  6 NOXA B PADLKDECAQLRRIGDKVNL  7 HRKSSAAQLTAARLKALGDELHQ  8 BNIP VVEGEKEVEALKKSADWVSD  9 PUMAEQWAREIGAQLRRMADDLNA 10 BMF HQAEVQIARKLQLIADQFHR 11 huBADNLWAAQRYGRELRRMSDEFVDSFK K 12 BADmut LWAAQRYGREARRMSDEFEGSFKGL 13

Mitochondria were probed to determine a cell's state using our panel ofsensitizer BH3-peptides, selective antagonists of antiapoptotic BCL-2family members. Mitochondria that are primed for death are dependent onantiapoptotic protein function to prevent MOMP, so that they releasecytochrome c when exposed to sensitizer BH3 peptides (See, FIG. 1, FIG.4A, FIG. 5C, and FIG. 6B). In contrast, unprimed cells do not releasecytochrome c when exposed to sensitizer BH3 peptides. Any cell fromwhich mitochondria can be isolated can therefore be so tested andcategorized as being primed or unprimed. Testing of mitochondriadirectly has the advantage of eliminating any contribution oftranscription, translation, or post-translational modification eventsthat might be triggered by transfection of peptide, protein, orexpression vector into a living cell. A “snapshot” of the apoptoticstate at a given time may be taken with minimal perturbation of theextant apoptotic machinery. In summary, the methods of the inventionallow capture if information about a fundamental aspect of cellularphysiology.

Importantly, mitochondrial behavior was correlated to whole cellbehavior in several models. Mitochondria were primed when cells wereenduring a physiologic challenge, and BH3 profiling revealed adependence on antiapoptotic proteins only when a cellular dependence wasalso demonstrated. As shown below in the EXAMPLES, FL5.12 cells andmitochondria became primed for death only after IL-3 withdrawal. For 2B4cells, cells and mitochondria were primed for death only afterdexamethasone treatment. For the primary BCL-2 dependent leukemia cells,the genomic instability, myc oncogene activation and checkpointviolation inherent to the cancer phenotype were sufficient to inducemitochondrial priming without further external intervention. The SCLCcell lines H164 and H1693 revealed a BCL-2 pattern of sensitivity to BH3profiling likewise are sensitive to the BCL-2 antagonist ABT-737. Ineach case, mitochondrial studies correctly diagnosed the cellulardependence on an antiapoptotic BCL-2 family member. Furthermore, theidentity of the individual family member could be decoded based on thepattern of mitochondrial sensitivity to our peptide panel. These resultsindicate that in some cells, like IL-3 replete FL5.12-BCL-2 cells, BCL-2overexpression provides extra antiapoptotic reserve. In others, like themurine leukemias, high levels of BCL-2 are present, but the BCL-2 is sohighly occupied by activator BH3 proteins that the cell has very poorantiapoptotic reserve, and is actually primed for death.

Not all cells are sensitive to antagonism of antiapoptotic proteins.Sensitive cells are “primed for death” with death signals carried by aselect subset of proapoptotic proteins of the BCL-2 family. Some cancercells may be tonically primed for death, and thus are selectivelysusceptible to agents that provoke or mimic sensitizer BH3-only domains.It has been postulated that inhibition of apoptosis is a requirement ofoncogenesis (Green and Evan, 2002; Hanahan and Weinberg, 2000). In whatmay be an attempt to meet this requirement, many types of cancer cellsoverexpress antiapoptotic BCL-2 family members. Understanding how theseproteins function is therefore critical to understanding how cancercells maintain survival. The methods of the present invention allows thesystematic investigation how antiapoptotic BCL-2 family members interactwith BH3-only family members to control mitochondrial outer membranepenneabilization (MOMP) and commitment to apoptosis. Antiapoptoticproteins show selective affinity for binding BH3 peptides derived fromBH3-only proteins. Furthermore, antagonism of antiapoptotic familymembers results in MOMP only when the antiapoptotic proteins are“primed” with activator BH3 proteins, validating the critical role ofactivator BH3 domains in activating BAX/BAK. In cell culture models,activator “priming” can be observed following experimentally-induceddeath signaling, and that such priming confers dependence onantiapoptotic family members. Remarkably, dependence on antiapoptoticBCL-2 family members can be captured functionally by the pattern ofmitochondrial sensitivity to sensitizer BH3 domains. Accordingly, theinvention features methods of determining the sensitivity of a cell to atherapeutic agent by identifying whether or not a cell is primed fordeath by determining the pattern of mitochondrial sensitivity to BH3domain peptides.

BH3 Profiling

In various methods, sensitivity of a cell to an agent is determined.Cell sensitivity is determined by contacting a cell or cellularcomponent (e.g., mitochondria) with a BH3 domain peptide. A cell issensitive to an agent if apoptosis is detected. Alternatively, cellsensitivity is determined by providing a test BH3 profile of the celland comparing the profile to a cancer cell BH3 profile. A similarity ofthe test profile and the control profile indicates that the cell issensitive to an agent. A BH3 profile is a pattern of sensitivity to BH3peptides of the cell. Sensitivity is indicated by apoptosis. A cancercell BH3 profile is a pattern of sensitivity to BH3 peptides in a cancercell whose responsiveness or lack thereof to a particular agent isknown. Optionally, the test BH3 profile is compared to more than onecancer cell BH3 profile. Thus, by comparing the test BH3 profile to thecontrol BH3 profile sensitivity to an agent is determined.

The cell or cellular component is a cancer cell or a cell that issuspected of being cancerous. The cell is permeabilized to permit theBH3 peptides access to the mitochondria. Cells are permeabilized bymethods known in the art. For example, the cell are permeabilized bycontacting the cell with digitonin. After the cell is permeabilized, thecells are treated with a potentiometric dye. Examples of potentiometricdyes include the green-fluorescent JC-1 probe(5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanineiodide) or dihydrorhodamine 123.

JC-1 is a lipophilic, cationic dye that enters mitochondria inproportion to the membrane potential JC-1 exists as a monomer in waterat low membrane potential (M). However, at higher potentials, JC-1 formsred-fluorescent “J-aggregates”. As a monomer the dye has anabsorption/emission maxima of 527 nm while at high membrane potentialthe emission maximum is 590 nm. Thus, ratio measurements of the emissionof this cyanine dye can be used as a sensitive measure of mitochondrialmembrane potential. The dye allows for a dual measurement of dyeconcentration that does not require the measurement of a nuclear orcytoplasmic reference values. Studies using isolated mitochondria haveshown that the 527 nm emission from monomeric JC-1 increases almostlinearly with membrane M potentials ranging from 46 to 182 mV, whereasthe 590 nm J-aggregate emission is less sensitive to M values lessnegative than 140 mv and is strongly sensitive to potential values inthe range of 140 to 182 mV (Di Lisa et al., 1995) Optical filtersdesigned for fluorescein and tetramethylrhodamine can be used toseparately visualize the monomer and J-aggregate forms, respectively.Alternatively, both forms can be observed simultaneously using astandard fluorescein longpass optical filter set.

Dihydrorhodamine 123 an uncharged, nonfluorescent agent that can beconverted by oxidation to the fluorescent laser dye rhodamine 123(R123).

The cell is from a subject known to or suspected of having cancer. Thesubject is preferably a mammal. The mammal is, e.g., a human, non-humanprimate, mouse, rat, dog, cat, horse, or cow. The subject has beenpreviously diagnosed as having cancer, and possibly has alreadyundergone treatment for cancer. Alternatively, the subject has not beenpreviously diagnosed as having cancer.

The agent is a therapeutic agent such as a chemotherapeutic agent. Forexample the agent is a mimetic of sensitizer BH3 domains or anantagonist of an anti-apoptotic protein. Apoptosis, i.e., cell death isidentified by know methods. For example, characteristics of apoptosisinclude the cell shrinks, develop bubble-like blebs on their surface,have the chromatin (DNA and protein) in their nucleus degraded, and havetheir mitochondria break down with the release of cytochrome c, loss ofmitochondrial membrane potential, break into small, membrane wrapped,fragments, or phosphatidylserine, which is normally hidden within theplasma membrane, is exposed on the surface of the cell.

The difference in the level apoptosis of a cell that has been contactedwith a BH3 peptide compared to a cell that has not been contacted with aBH3 peptide is statistically significant. By statistically significantis meant that the alteration is greater than what might be expected tohappen by chance alone. Statistical significance is determined by methodknown in the art. For example statistical significance is determined byp-value. The p-value is a measure of probability that a differencebetween groups during an experiment happened by chance.(P(z>Z_(observed))). For example, a p-value of 0.01 means that there isa 1 in 100 chance the result occurred by chance. The lower the p-value,the more likely it is that the difference between groups was caused bytreatment. An alteration is statistically significant if the p-value isor less than 0.05. Preferably, the p-value is 0.04, 0.03, 0.02, 0.01,0.005, 0.001 or less.

The invention also includes a profile of a pattern of mitochondrialsensitivity to BH3 sensitizer peptides taken from one or more subjectswho have cancer.

BH3 Domain Peptides

A BH3 domain peptide is less than 195 amino acids in length, e.g., lessthan or equal to 150, 100, 75, 50, 35, 25 or 15 amino acid in length.For example a BH3 peptide includes the sequence of SEQ ID NO: 1-13 shownin Table 1.

A BH3 domain peptide include a peptide which includes (in whole or inpart) the sequence NH₂— XXXXXXIAXXLXXXGDXXXX —COOH (SEQ ID NO: 14) orNH₂— XXXXXXXXXXLXXXXDXXXX —COOH (SEQ ID NO: 15). As used herein X may beany amino acid. Alternatively, the BH3 domain peptides include at least5, 6, 7, 8, 9, 15 or more amino acids of SEQ ID NO: 14 or SEQ ID NO:15).

Optionally, the BH3 domain peptide is attached to transduction domain. Atransduction domain compound that directs a peptide in which it ispresent to a desired cellular destination Thus, the transduction domaincan direct the peptide across the plasma membrane, e.g., from outsidethe cell, through the plasma membrane, and into the cytoplasm.Alternatively, or in addition, the transduction domain can direct thepeptide to a desired location within the cell, e.g., the nucleus, theribosome, the ER, mitochondria, a lysosome, or peroxisome.

In some embodiments, the transduction domain is derived from a knownmembranetranslocating sequence. Alternatively, transduction domain is acompound that is known to facilitate membrane uptake such aspolyethylene glycol, cholesterol moieties, octanoic acid and decanoicacid.

For example, the trafficking peptide may include sequences from thehuman immunodeficiency virus (HIV) 1 TAT protein. This protein isdescribed in, e.g., U.S. Pat. Nos. 5,804,604 and 5,674,980, eachincorporated herein by reference. The BH3 domain peptide is linked tosome or all of the entire 86 amino acids that make up the TAT protein.For example, a functionally effective fragment or portion of a TATprotein that has fewer than 86 amino acids, which exhibits uptake intocells can be used. See e.g., Vives et al., J. Biol. Chem.,272(25):16010-17 (1997), incorporated herein by reference in itsentirety. A TAT peptide that includes the region that mediates entry anduptake into cells can be further defined using known techniques. See,e.g., Franked et al., Proc. Natl. Acad. Sci, USA 86: 7397-7401 (1989).Other sources for translocating sequences include, e.g., VP22 (describedin, e.g., WO 97/05265; Elliott and O'Hare, Cell 88: 223-233 (1997)),Drosophila Antennapedia (Antp) homeotic transcription factor, HSV,poly-arginine, poly lysine, or non-viral proteins (Jackson et al, Proc.Natl. Acad. Sci. USA 89: 10691-10695 (1992)).

The transduction domain may be linked either to the N-terminal or theC-terminal end of BH3 domain peptide. A hinge of two proline residuesmay be added between the transduction domain and BH3 domain peptide tocreate the full fusion peptide. Optionally, the transduction domain islinked to the BH3 domain peptide in such a way that the transductiondomain is released from the BH3 domain peptide upon entry into the cellor cellular component.

The transduction domain can be a single (i.e., continuous) amino acidsequence present in the translocating protein. Alternatively it can betwo or more amino acid sequences, which are present in protein, but inthe naturally-occurring protein are separated by other amino acidsequences.

The amino acid sequence of naturally-occurring translocation protein canbe modified, for example, by addition, deletion and/or substitution ofat least one amino acid present in the naturally-occurring protein, toproduce modified protein. Modified translocation proteins with increasedor decreased stability can be produced using known techniques. In someembodiments translocation proteins or peptides include amino acidsequences that are substantially similar, although not identical, tothat of naturally-occurring protein or portions thereof. In addition,cholesterol or other lipid derivatives can be added to translocationprotein to produce a modified protein having increased membranesolubility.

The BH3 domain peptide and the transduction domain can be linked bychemical coupling in any suitable manner known in the art. Many knownchemical cross-linking methods are non-specific, i.e.; they do notdirect the point of coupling to any particular site on the transportpolypeptide or cargo macromolecule. As a result, use of non-specificcross-linking agents may attack functional sites or sterically blockactive sites, rendering the conjugated proteins biologically inactive.

One way to increasing coupling specificity is to directly chemicalcoupling to a functional group found only once or a few times in one orboth of the polypeptides to be crosslinked. For example, in manyproteins, cysteine, which is the only protein amino acid containing athiol group, occurs only a few times. Also, for example, if apolypeptide contains no lysine residues, a cross-linking reagentspecific for primary amines will be selective for the amino terminus ofthat polypeptide. Successful utilization of this approach to increasecoupling specificity requires that the polypeptide have the suitablyrare and reactive residues in areas of the molecule that may be alteredwithout loss of the molecule's biological activity.

Cysteine residues may be replaced when they occur in parts of apolypeptide sequence where their participation in a cross-linkingreaction would otherwise likely interfere with biological activity. Whena cysteine residue is replaced, it is typically desirable to minimizeresulting changes in polypeptide folding. Changes in polypeptide foldingare minimized when the replacement is chemically and sterically similarto cysteine. For these reasons, serine is preferred as a replacement forcysteine. As demonstrated in the examples below, a cysteine residue maybe introduced into a polypeptide's amino acid sequence for cross-linkingpurposes. When a cysteine residue is introduced, introduction at or nearthe amino or carboxy terminus is preferred. Conventional methods areavailable for such amino acid sequence modifications, whether thepolypeptide of interest is produced by chemical synthesis or expressionof recombinant DNA.

Coupling of the two constituents can be accomplished via a coupling orconjugating agent. There are several intermolecular cross-linkingreagents which can be utilized, See for example, Means and Feeney,CHEMICAL MODIFICATION OF PROTEINS, Holden-Day, 1974, pp. 39-43. Amongthese reagents are, for example, J-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) or N,N′-(1,3-pbenylene) bismaleimide (both of whichare highly specific for sulfhydryl groups and form irreversiblelinkages); N,N′-ethylene-bis-(iodoacetamide) or other such reagenthaving 6 to 11 carbon methylene bridges (which relatively specific forsulfhydryl groups); and 1,5-difluoro-2,4-dinitrobenzene (which formsirreversible linkages with amino and tyrosine groups). Othercross-linking reagents useful for this purpose include:p,p′-difluoro-m,m′dinitrodiphenylsulfone (which forms irreversiblecross-linkages with amino and phenolic groups); dimethyl adipimidate(which is specific for amino groups); phenol-1,4-disulfonylchloride(which reacts principally with amino groups); hexamethylenediisocyanateor diisothiocyanate, or azophenyl-p-diisocyanate (which reactsprincipally with amino groups); glutaraldehyde (which reacts withseveral different side chains) and disdiazobenzidine (which reactsprimarily with tyrosine and histidine).

Cross-linking reagents may be homobifunctional, i.e., having twofunctional groups that undergo the same reaction. A preferredhomobifunctional cross-linking reagent is bismaleimidohexanc (“BMH”).BMH contains two maleimide functional groups, which react specificallywith sulfhydryl-containing compounds under mild conditions (pH 6.5-7.7).The two maleimide groups are connected by a hydrocarbon chain.Therefore, BMH is useful for irreversible cross-linking of polypeptidesthat contain cysteine residues.

Cross-linking reagents may also be heterobifunctional.Heterobifunctional cross-linking agents have two different functionalgroups, for example an amine-reactive group and a thiol-reactive group,that will cross-link two proteins having free amines and thiols,respectively. Examples of heterobifunctional cross-linking agents aresuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (“SMCC”),m-maleimidobenzoyl-N-hydroxysuccinimide ester (“MBS”), and succinimide4-(p-maleimidophenyl) butyrate (“SMPB”), an extended chain analog ofMBS. The succinimidyl group of these cross-linkers reacts with a primaryamine, and the thiol-reactive maleimide forms a covalent bond with thethiol of a cysteine residue.

Cross-linking reagents often have low solubility in water. A hydrophilicmoiety, such as a sulfonate group, may be added to the cross-linkingreagent to improve its water solubility. Sulfo-MBS and sulfo-SMCC areexamples of cross-linking reagents modified for water solubility.

Many cross-linking reagents yield a conjugate that is essentiallynon-cleavable under cellular conditions. However, some cross-linkingreagents contain a covalent bond, such as a disulfide, that is cleavableunder cellular conditions. For example, Traut's reagent, dithiobis(succinimidylpropionate) (“DSP”), and N-succinimidyl 3-(2-pyridyldithio)propionate (“SPDP”) are well-known cleavable cross-linkers. The use of acleavable cross-linking reagent permits the cargo moiety to separatefrom the transport polypeptide after delivery into the target cell.Direct disulfide linkage may also be useful.

Numerous cross-linking reagents, including the ones discussed above, arecommercially available. Detailed instructions for their use are readilyavailable from the commercial suppliers. A general reference on proteincross-linking and conjugate preparation is: Wong, CHEMISTRY OF PROTEINCONJUGATION AND CROSS-LINKING, CRC Press (1991).

Chemical cross-linking may include the use of spacer arms. Spacer armsprovide intramolecular flexibility or adjust intramolecular distancesbetween conjugated moieties and thereby may help preserve biologicalactivity. A spacer arm may be in the form of a polypeptide moiety thatincludes spacer amino acids, e.g. proline. Alternatively, a spacer armmay be part of the cross-linking reagent, such as in “long-chain SPDP”(Pierce Chem. Co., Rockford, Ill., cat. No. 21651 H).

The BH3 domain peptides and/or the transduction domain peptides can bepolymers of L-amino acids, D-amino acids, or a combination of both. Forexample, in various embodiments, the peptides are D retro-inversopeptides. The term “retro-inverso isomer” refers to an isomer of alinear peptide in which the direction of the sequence is reversed andthe chirality of each amino acid residue is inverted. See, e.g., Jamesonet al., Nature, 368, 744-746 (1994); Brady et al., Nature, 368, 692-693(1994). The net result of combining D-enantiomers and reverse synthesisis that the positions of carbonyl and amino groups in each amide bondare exchanged, while the position of the side-chain groups at each alphacarbon is preserved. Unless specifically stated otherwise, it ispresumed that any given L-amino acid sequence of the invention may bemade into a D retro-inverso peptide by synthesizing a reverse of thesequence for the corresponding native L-amino acid sequence.

Alternatively, the BH3 domain peptides and/or the transduction domainpeptides are cyclic peptides. Cyclic peptides are prepared by methodsknown in the art. For example, macrocyclization is often accomplished byforming an amide bond between the peptide N- and C-termini, between aside chain and the N- or C-terminus [e.g., with K₃Fe(CN)₆ at pH 8.5](Samson et al., Endocrinology, 137: 5182-5185 (1996)), or between twoamino acid side chains. See, e.g., DeGrado, Adv Protein Chem, 39: 51-124(1988).

BH3 domain peptides and/or the transduction domain peptides are easilyprepared using modern cloning techniques, or may be synthesized by solidstate methods or by site-directed mutagenesis. A domain BH3 peptideand/or the transduction domain peptides may include dominant negativeforms of a polypeptide. In one embodiment, native BH3 domain peptidesand/or transduction domain peptides can be isolated from cells or tissuesources by an appropriate purification scheme using standard proteinpurification techniques. In another embodiment, BH3 domain polypeptidesand/or transduction domain peptides are produced by recombinant DNAtechniques. Alternative to recombinant expression, BH3 domain peptidesand/or transduction domain peptides can be synthesized chemically usingstandard peptide synthesis techniques.

An “isolated” or “purified” protein or biologically active portionthereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which the BH3domain peptide is derived, or substantially free from chemicalprecursors or other chemicals when chemically synthesized. The language“substantially free of cellular material” includes preparations of BH3peptides and/or transduction domain peptides in which the protein isseparated from cellular components of the cells from which it isisolated or recombinantly produced. In one embodiment, the language“substantially free of cellular material” includes preparations of BH3domain peptides and/or the transduction domain peptides having less thanabout 30% (by dry weight) of non-BH3 domain peptide and/ornon-transduction domain peptides (also referred to herein as a“contaminating protein”), more preferably less than about 20% of non-BH3peptide and/or non-transduction domain peptides, still more preferablyless than about 10% of non-BH3 peptide and/or non-transduction domainpeptides, and most preferably less than about 5% non-BH3 domain peptideand/or non-transduction domain peptides. When the BH3 domain peptideand/or the transduction domain peptides or biologically active portionthereof is recombinantly produced, it is also preferably substantiallyfree of culture medium, i.e., culture medium represents less than about20%, more preferably less than about 10%, and most preferably less thanabout 5% of the volume of the protein preparation.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of BH3 domain peptides and/or thetransduction domain peptides in which the protein is separated fromchemical precursors or other chemicals that arc involved in thesynthesis of the protein. In one embodiment, the language “substantiallyfree of chemical precursors or other chemicals” includes preparations ofBH3 domain peptides and/or transduction domain peptides having less thanabout 30% (by dry weight) of chemical precursors or non-BH3 domainpeptide and/or non-transduction domain peptides chemicals, morepreferably less than about 20% chemical precursors or non-BH3 domainpeptide and/or non-transduction domain peptides chemicals, still morepreferably less than about 10% chemical precursors or non-BH3 domainpeptide chemicals, and most preferably less than about 5% chemicalprecursors or non-BH3 domain peptide and/or non-transduction domainpeptides chemicals.

The term “biologically equivalent” is intended to mean that thecompositions of the present invention are capable of demonstrating someor all of the same apoptosis modulating effects, i.e., release ofcytochrome c or BAK oligomerization although not necessarily to the samedegree as the BH3 domain polypeptide deduced from sequences identifiedfrom cDNA libraries of human, rat or mouse origin or produced fromrecombinant expression symptoms.

Percent conservation is calculated from the above alignment by addingthe percentage of identical residues to the percentage of positions atwhich the two residues represent a conservative substitution (defined ashaving a log odds value of greater than or equal to 0.3 in the PAM250residue weight table). Conservation is referenced to sequences asindicated above for identity comparisons. Conservative amino acidchanges satisfying this requirement are: R-K; E-D, Y-F, L-M; V-I, Q-H.

BH3 domain peptides can also include derivatives of BH3 domain peptideswhich are intended to include hybrid and modified forms of BH3 domainpeptides including fusion proteins and BH3 domain peptide fragments andhybrid and modified forms in which certain amino acids have been deletedor replaced and modifications such as where one or more amino acids havebeen changed to a modified amino acid or unusual amino acid andmodifications such as glycosylation so long as the hybrid or modifiedform retains the biological activity of BH3 domain peptides. Byretaining the biological activity, it is meant that cell death isinduced by the BH3 polypeptide, although not necessarily at the samelevel of potency as that of the naturally-occurring BH3 domainpolypeptide identified for human or mouse and that can be produced, forexample, recombinantly. The terms induced and stimulated are usedinterchangeably throughout the specification.

Preferred variants are those that have conservative amino acidsubstitutions made at one or more predicted non-essential amino acidresidues. A “conservative amino acid substitution” is one in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Thus, a predicted nonessential amino acid residue in a BH3domain polypeptide is replaced with another amino acid residue from thesame side chain family. Alternatively, in another embodiment, mutationscan be introduced randomly along all or part of a BH3 coding sequence,such as by saturation mutagenesis, and the resultant mutants can bescreened to identify mutants that retain activity.

Also included within the meaning of substantially homologous is any BH3domain peptide which may be isolated by virtue of cross-reactivity withantibodies to the BH3 domain peptide described herein or whose encodingnucleotide sequences including genomic DNA, mRNA or cDNA may be isolatedthrough hybridization with the complementary sequence of genomic orsubgenomic nucleotide sequences or cDNA of the BH3 domain peptidesherein or fragments thereof.

The invention will be further illustrated in the following non-limitingexamples.

EXAMPLE 1 General Methods Reagents

ABT-737 and its negative control enantiomer which has lower affinity forBCL-2 family members were obtained from Abbott Laboratories(OltersdorfT, 2005).

GST-Pulldown

10 ug GST-BCL-w (or BH3 binding-defective R96P point mutant) wereincubated with glutathione-agarose beads for one hour at 4° C. inbinding buffer (140 mM NaCl, 10 mM Tris pH7.4). Beads were rinsed andincubated with approximately 0.2 ug tBID for 1 hr at 4° C. Beads werewashed again and incubated with peptides for 1 hour at 4° C. tBTDprotein was eluted from beads with 50 mM glutathione and loaded on adenaturing NuPAGE gel.

Cytochrome c Release

Mitochondria were purified from liver and FL5.12cells as previouslydescribed (Letai et al., 2002). Mitochondria were purified from leukemiacells and 2B4 cells as previously described for FL5.12 cells.Mitochondria were incubated with treatments for 45 (mouse livermitochondria) or 35 minutes (FL5.12, 2B4, and leukemic mitochondria).Release of cytochrome c was determined by a comparison of cytochrome cin the pellet and supernatant following treatment, quantitated by ELISA(R&D systems). When results of multiple experiments were averaged,results from solvent-only (DMSO) treatments values were subtracted fromeach, so that 0 release reflects that observed in solvent-onlytreatments.

Alternatively, mitochondria were purified from freshly isolated CLLcells and cell lines by mechanical disruption followed by differentialcentrifugation, as previously described (Letai et al., 2002).Mitochondrial suspensions were made at 0.5 mg protein/ml, except for thecase of ABT-737 and negative control enantiomer treatments, where 0.1mg/ml was used. Release of cytochrome c was determined by a comparisonof cytochrome c in the pellet and supernatant quantitated by ELISA (R&Dsystems).

Peptides

Peptides were synthesized by Tufts University Core Facility and purifiedby HPLC. Identity was confirmed by mass spectrometry. Stock solutionswere made in DMSO. Peptides used for fluorescence polarization weresynthesized with an N-terminus fluorescein tag and a 6-aminohexanoicacid linker. Sequences were taken from published sequences of murine BAD(LWAAQRYGRELRRMSDEFEGSFKGL; SEQ ID NO: 4) (Kelekar et al., 1997), BADmu(LWAAQRYGREARRMSDEFEGSFKGL; SEQ ID NO: 13, note L→A, a point mutationabrogating binding to BCL-2) (Zha et al., 1997), NOXA A(AELPPEFAAQLRKIGDKVYC; SEQ ID NO: 6) (Oda et al., 2000), BMF(HQAEVQIARKLQLIADQFHR; SEQ ID NO: 11) (Puthalakath et al., 2001) andhuman BID (EDIIRNIARHLAQVGDSMDR; SEQ ID NO: 1) (Wang et al., 1996), BIM(MRPEIWIAQELRRIGDEFNA; SEQ ID NO: 2) (O'Connor et al., 1998), BIK(MEGSDALALRLACIGDEMDV; SEQ ID NO: 5) (Boyd et al., 1995), BNIP3-α(VVEGEKEVEALKKSADWVSD; SEQ ID NO: 9) (Yasuda et al., 1999), hari-kiri(HRK) (SSAAQLTAARLKALGDELHQ; SEQ ID NO: 8) (Inohara et al., 1997) andPUMA (EQWAREIGAQLRRMADDLNA; SEQ ID NO: 10) (Nakano and Vousden, 2001).

Recombinant Proteins

Antiapoptotic proteins were expressed in bacteria and affinity purifiedusing glutathioneagarose (for GST-linked proteins) as previouslydescribed (Letai et al., 2002) or by nickel-NTA agarose beads (forHis-tagged MCL-1) according to manufacturer protocol (Qiagen). Selectedsamples were additionally purified by anion exchange FPLC to obtainsufficient purity as judged by Coomassie staining of a denaturing gel.In each case, the C-terminus transmembrane domain was truncated tomaintain solubility in aqueous solution. For binding assays, GST-linkedproteins were used for BCL-2, BCL-XL, BCL-w and BFL-1; His-tagged MCL-1was used. For the mitochondrial assays, the same proteins were usedexcept for MCL-1, where a GST-linked protein was used. Constructsexpressing GST-MCL-1, GST-BFL-1, and GST-BCL-w were kind gifts of TilmanOltersdorf; His-tagged MCL-1 construct was a kind gift of Ruth Craig.The human sequence was used for each. Recombinant tBID was made aspreviously described; it contained double cysteine to serinesubstitutions which maintains wild-type ability to induce cytochrome crelease(Oh et al., 2005).

Fluorescence Polarization Binding Assays

Binding assays were performed using fluorescence polarization aspreviously described (Letai et al., 2002). A minimum of threeindependent experiments were used to determine each dissociationconstant. For BIM BH3 displacement assays, 25 nM fluorescein-linked BIMBH3 peptide was bound to 0.5 uM GST-BCL-2 or 0.1 uM GST-MCL-1 in bindingbuffer. NOXA or BAD BH3 peptides were then titrated and displacement ofBIM BH3 monitored by loss of fluorescence polarization.

Immunoprecipitation

Cell lysates (250 ug) were incubated with 6C8 hamster anti-human BCL-2antibody (3 ug) for at least 1 hour at room temperature in 1% CHAPSbuffer (5 mM sodium phosphate pH 7.4; 2.5 mM EDTA; 100 mM sodiumchloride; 1% w/v CHAPS, in the presence of protease inhibitors (Completetablets; Roche)). Protein A-sepharose beads (Sigma) were added toprecipitate complexes containing BCL-2. The beads were mixed withloading buffer prior to loading supernatant onto gel. For FLAG-MCL-1immunoprecipitation, 2B4 cells were treated with 100 nM dexamethasonefor 24 hours and lysed in 1% CHAPS buffer. 250 ug protein lysate wasincubated with anti-FLAG antibody-conjugated agarose beads (Sigma) for 1hour at 4° C. Protein was eluted from washed beads with 2.5 ug FLAGpeptide. Eluant was loaded onto denaturing gel for electrophoresis.

Alternatively, Cell lysates (250 μg) were incubated with 6C8 hamsteranti-human BCL-2 antibody (3 μg) for at least 1 hour at 4° C. in 0.1%Triton-X100 buffer. Protein A-sepharose beads (Sigma) were added toprecipitate complexes containing BCL-2. The beads were mixed withloading buffer prior to loading supernatant onto a 100% Bis-Trispolyacrylamide gel for analysis. For displacement reactions, 50 μg oflysate were incubated with 3 μg 6C8 BCL-2 antibody for at least 1 hourat 4° C. in 0.1% Triton-X100 buffer or CHAPS buffer. Protein A-sepharosebeads were added and incubated for 1 hour. Then the beads were pelletedand washed 3 times and resuspended in HE buffer (1 mM EDTA and 10 mMHEPES, pH 7.4, as in (Chipuk et al. 2004)). 1 μM ABT-737, 1 μM negativecontrol enantiomer, or DMSO was added to the tube, incubated overnight;the supernatant was loaded onto a 10% Bis-Tris polyacrylamide gel(Invitrogen) for analysis.

Immunoblots

Protein lysates were obtained by cell lysis in 1% CHAPS buffer. Proteinsamples were size fractionated on NuPAGE 10% Bis-Tris polyacrylamidegels (lnvitrogen). Antibodies were used to detect the following proteinson membrane: BIM (Calbiochem, 22-40); BCL-2 (Pharmingen, /100); PUMA(Prosci, NT); rabbit polyclonal anti-murine BID (Wang et al., 1996); BAK(Upstate, NT); BAX (Santa Cruz, N-20); Actin (Chemicon, MAB1501); CD-40(Pharmingen, HM40-3); MCL-1 (Rockland). BAX oligomerization performed aspreviously described (Letai et al., 2002).

Alternatively, protein lysates were obtained by cell lysis inTriton-X100 (142.5 mM NaCl, 5 mM MgCl2, 10 mM HEPES, 1 mM EGTA, 0.1%Triton-X100 (Sigma)), RIPA (150 mM NaCl, 2 mM EDTA, 0.1 M Na2HPO4 pH7.2, 0.2 mM NaVO4, 50 mM NaF, 1% sodium deoxycholate, 0.1% SDS, and%NP-40(Sigma)) or CHAPS (100 mM NaCl, 5 mM NaP04, 2.5 mM EDT A, 1% CHAPS(Sigma)) buffer supplemented with a Complete protease inhibitor cocktailtablet (Roche). Protein samples were electrophoretically separated onNuPAGE 10% Bis-Tris polyacrylamide gels (Invitrogen). Antibodies wereused to detect the following proteins on membrane: BIM (Calbiochem 22-40or Abgent BH3 domain); BCL-2 (Pharmingen, /100); MCL-1 (Chemicon,RC-13).

Annexin-V Assay

Cells were stained with fluorescent conjugates of Annexin-V (BioVision)and propidium iodide (PI) and analyzed on a FACSCalibur machine(Becton-Dickinson).

Isolation and Short-Term Culture of Human Cells

15 ml of blood in heparin treated tubes was obtained from each anonymousCLL patient and processed without freezing. Equal volume of media (RPMImedium, 10% human bovine serum, supplemented with 10 μm/ml insulin and10 mg/ml transferrin) was mixed with each sample and CLL cells isolatedby centrifugation through Ficoll-PAQUE Plus (Amersham). Cells werewashed twice in media and cultured at a density of 2.0×106 cells/ml forup to 48 hours. Samples for FIG. 11A were obtained from 24 consecutivepatients identified with WBC >50,000/μl. All guidelines and regulationswere followed in accordance with IRB protocols #99-224 (Dana-FarberCancer Institute). Normal PBMC's were obtained from tubing discardedfollowing platelet donation by anonymous normal donors and processed asabove, except cultured without insulin and transferrin.

CLL Clinical Criteria

FISH analysis was performed by the Brigham and Women's Hospitalcytogenetics laboratory using a CLL panel of multicolor probe sets(Vysis, Inc.) (Dohner et al., 2000). CLL cells were processed and IgVHand ZAP70 status were determined by the CLL Research Consortium Tissuecore using previously established methods (Rassenti et al. 2004).Somatic hypermutation in the IgVH locus was classified as absentwhen >98% homology to germline was measured. Patient samples wereclassified as ZAP70 positive when >20% cells were positive; CD38positive when >30% cells were positive. Multiple Myeloma Cell cultureLP1 and L363 cells (kind gift from Ruben Carrasco) were cultured inIscove's modified Dulbecco's medium with 10% fetal bovine serum.

Cell Culture

FL5.12 cells were cultured as described previously in Iscove's modifiedDulbecco's medium, 10% fetal bovine serum, 1000 ug/ml G418 with orwithout IL-3 provided by 10% WEHI-3B supplement (supernatant of IL-3secreting WEHI-3B cells). FL5.12 cells were stably transfected with avector containing a neomycin resistance construct and either human BCL-2cDNA (FL5.12-BCL-2) or no insert (wt). 2B4 cells were cultured in RPMI1640 supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100ug/ml streptomycin, 10 uM non-essential amino acids and 8 ul/Lbetamercaptoethanol. Stably transfected 2B4 cells were isolated aftertransfection with pLZR-GFP retroviral vector or vector with Flag-Mc1-1(kind gift from Joe Opferman).

Caspase Inhibition Experiment

10⁶ cells/mL Neo or BCL-2 FL5.12 cells were plated in the mediacontaining IL-3, or 2.0×10⁶ cells/mL cells were washed twice with 1× PBSand plated in media without IL-3 for 24 hours. Cells receiving caspaseinhibitor were incubated with 200 μM ZVAD.fmk (Calbiochem) for 1 hourprior to any additional treatment. Cells were treated with 1 μM ABT-737or NCE for 30 minutes, 1, 2, 3 or 4 hours as indicated and then stainedwith Annexin-V/PI to assess apoptotic status. For protein analysis,cells were harvested, washed with 1× PBS twice and lysates made in 1%CHAPS buffer. lOug was loaded onto a 10% Bis-Tris protein gel. Resultingimmunoblots were probed with anti-PARP antibody (BioVision), whichrecognizes both cleaved and uncleaved PARP protein.

BAX oligomerization

10⁷ freshly isolated CLL cells were incubated with DMSO, 10 nM, 100 nM,or 1 μM ABT-737 or negative control enantiomer for 4 hours, % deadassessed by Annexin-V staining and FACS analysis, then treated with 0.3%saponin and 10 μM BMH for 30 minutes on ice. Cells were then lysed,loaded onto a 10% BISTris polyacrlyamide gel, transferred, andimmunoblotted for BAX.

Mice

Leukemia prone mice were generated as previously described (Letai,2004). Mouse experimental protocols conform to the relevant regulatorystandards and were approved by the Dana-Farber Cancer Institute AnimalCare and Use Committee.

Statistical Analyses

Experimental replicates were performed using lysates or mitochondriafrom different CLL samples. In main body, where a P value is given, itwas obtained by using a two-tailed Students t-test, and P <0.05 wasconsidered statistically significant. GraphPad Prism software was usedto determine EC50 values by non-linear dose-response curve fitting andto perform Mann-Whitney nonparametric testing in Table 5.

EXAMPLE 2 Antiapoptotic Proteins Demonstrate Distinct Profiles ofBinding Sensitizer BH3 Peptides

To determine selectivity in interactions among antiapoptotic BCL-2family members and BH3 domains of BH3-only proteins, fluorescencepolarization binding assays (FPA) were used. Antiapoptotic proteinsBCL-2, BCL-XL, MCL-1, BCL-w, and BFL-1 were purified from transfectedbacteria as GST fusion proteins. BH3-domains were synthesized as20-25-mers as shown in Table 2A. Oligopeptides used for FPA were taggedwith an N-terminal FITC moiety. Table 2B quantitates binding bydissociation constants.

TABLE 2A BID EDIIRNIARHLAQVGDSMDR (SEQ ID NO: 1) BIMMRPEIWIAQELRRIGDEFNA (SEQ ID NO: 2) BIDmut EDIIRNIARHAAQVGASMDR(SEQ ID NO: 3) BAD LWAAQRYGRELRRMSDEFEGSFKGL (SEQ ID NO: 4) BIKMEGSDALALRLACIGDEMDV (SEQ ID NO: 5) NOXA A AELPPEFAAQLRKIGDKVYC(SEQ ID NO: 6) NOXA B PADLKDECAQLRRIGDKVNL (SEQ ID NO: 7) HRKSSAAQLTAARLKALGDELHQ (SEQ ID NO: 8) BNIP VVEGEKEVEALKKSADWVSD(SEQ ID NO: 9) PUMA EQWAREIGAQLRRMADDLNA (SEQ ID NO: 10) BMFHQAEVQIARKLQLIADQFHR (SEQ ID NO: 11)

TABLE 2B BID BIM BIDmut BAD BIK NOXA A NOXA B HRK BNIP PUMA BMF BCL-2 66(6) <10 — 11 (3) 151 (2) — — — — 18 (1) 24 (1) BCL-XL 12 (9) <10 — <10 10 (2) — — 92 (11) — <10 <10 BCL-w <10 38 (7) — 60 (19)  17 (12) — — —— 25 (12) 11 (3) MCL-1 <10 <10 — — 109 (33) 19 (2) 28 (3) — — <10 23 (2)BFL-1 53 (3) 73 (3) — — — — — — — 59 (11) —

It is immediately notable that the antiapoptotic family members may bedistinguished from each other based on affinity for individual BH3domains. For instance, BCL-XL may be distinguished from BCL-2 and BCL-wby its much greater affinity for HRK BH3. Otherwise, though there arequantitative distinctions among binding patterns of BCL-2, BCL-XL andBCL-w, the qualitative binding patterns are quite similar, suggestingsimilarity in the hydrophobic binding pockets of these three molecules.

In contrast with this group, MCL-1 does not bind BAD BH3, in agreementwith data generated by pull-down (Opferman et al., 2003), yeasttwo-hybrid (Leo et al., 1999); and surface plasmon resonance (Chen etal., 2005) assays. Murine NOXA is unique among the known BH3-onlyproteins in that it possesses two 5 putative BH3 domains (Oda et al.,2000). It is notable that while the other four proteins interact withneither of the NOXA BH3 domains tested, MCL-1 interacts with both. Thissuggests that the interaction between NOXA and MCL-1 is indeedbiologically significant. The ability to bind both BH3 domains suggeststhe possibility of novel multimeric interactions between MCL-1 andmurine NOXA, or alternatively differential control over exposure of thetwo BH3 domains in NOXA.

Also distinct is BFL-1. While it binds BID and BIM, it binds only PUMAamong the sensitizers tested. It is also notable that the activators BIDand BIM BH3 are bound by all of the antiapoptotics tested,distinguishing them from the sensitizers which, except PUMA, show a moreselective pattern of binding. Of additional note is that the BH3 domainobtained from BNIP-3a binds to none of the proteins tested, and does notactivate BAX or BAK. While the possibility that BNIP BH3 interacts withan untested multi-domain pro- or antiapoptotic BCL-2 family membercannot be excluded, it is also possible that BNIP-3a does not functionas a BH3-family member at all (Ray et al., 2000).

EXAMPLE 3 Dependence on Individual Antiapoptotic Proteins may be Deducedby Pattern of Sensitivity to Sensitizer BH3 Peptides; Inhibition ofAntiapoptotic Protein is Insufficient For MOMP Unless Activator TBID isPresent

Previous results have shown that the BH3 domains of BID and BIM possessthe ability to induce BAX and BAK oligomerization and cytochrome crelease in a purified mitochondrial system (Letai et al., 2002). Thisclass is referred to as the BIB domain “activators.” BH3 domains fromBAD and BIK (termed “sensitizers”) were unable to induce cytochrome crelease on their own. However, when an activator was bound andsequestered by BCL-2, preventing interaction of the activator with BAXor BAK, sensitizers could provoke mitochondrial apoptosis bycompetitively inhibiting BCL-2′s binding of the activator, freeing theactivator to oligomerize BAX or BAK and induce cytochrome c release.Thus, the two sensitizer BH3 domains were shown to be antagonists ofBCL-2 antiapoptotic function. The ability to antagonize BCL-2 functioncorrelated with high-affinity binding to BCL-2.

In Table 2B above, the expanded range of BH3 domains tested in thepresent study demonstrate distinct patterns of binding to antiapoptoticproteins. To test if selective binding corresponded to ability ofindividual BH3 domains to selectively antagonize antiapoptotic function,a purified mitochondrial system was constructed in which the criticalapoptosis decision making molecular machinery was reconstituted. For theactivator function, caspase-8 cleaved BID protein, tBID, was used. tBIDis an archetypical activator protein, capable of inducing BAX/BAKoligomerization and cytochrome c release in purified mitochondria (Weiet al., 2000) and synthetic liposomes (Kuwana et al., 2005; Kuwana etal., 2002). tBID's induction of cytochrome c release and apoptosisrequires BAX or BAK (Cheng et al., 2001; Wei et al., 2001). Themultidomain proapoptotic function was provided by the BAK which residesin mouse liver Mitochondria; mouse liver mitochondria contain nodetectable BAX protein (Letai et al., 2002). The dominant antiapoptoticfunction was provided by one of the five different recombinantantiapoptotic proteins used in the binding assays. BH3 peptides providedthe sensitizer function.

As cytochrome c release is the readout for the system, it was importantto test whether the peptides by themselves release cytochrome c in mouseliver mitochondria like activators BID or BIM BH3 (Letai et al., 2002).FIG. 1A is a confirmatory assay that shows none of the sensitizer BH3peptides by themselves can induce cytochrome c release significantlyabove background, even at concentrations 10-fold higher than those usedin FIGS. 1B-1F. While this has previously been shown for BAD, BIK, NOXAA, and NOXA B BH3's, this is a novel finding for the HRK, BNIP, PUMA andBMF BH3 domains.

In each of the subsequent panels, cytochrome c release by tBID isdemonstrated, followed by inhibition of cytochrome c release by additionof either BCL-2 (FIG. 1B), BCL-XL (FIG. 1C), BCL-w (FIG. 1D), MCL-1(FIG. 1E), or BFL-1 (FIG. 1F). The ability of the panel of BH3 domainsto antagonize antiapoptotic protection as measured by cytochrome crelease was determined. Remarkably, in each case, the ability toantagonize antiapoptotic function maps to the binding specificities inTable 2B. This is important confirmation that the binding patternelucidated in Table 2B corresponds to biological function.

It is important to emphasize that treatment with sensitizer peptidesalone, even those that bind and antagonize all the antiapoptoticstested, such as PUMA BH3, or the combination of NOXA and BAD BH3 isinsufficient to cause cytochrome c release (FIG. 1A). Furthermore, whenthe panel of sensitizer BH3 peptides was tested in the presence of theantiapoptotic protein BCL-XL, there was still no cytochrome c release,formally ruling out the possibility that the BH3 peptides were somehowdirectly converting antiapoptotic proteins to a proapoptotic function(FIG. 2). To induce MOMP and cytochrome c release, there appears to bean absolute requirement for an activator function, here provided by thetBID protein.

These data critically demonstrate that the panel of peptides candetermine whether a mitochondrion depends on an antiapoptotic protein tomaintain integrity. Furthermore, the identity of the criticalantiapoptotic protein can be deduced based on the pattern of sensitivityto the panel of sensitizer BH3 peptides. This strategy is termed BH3profiling.

EXAMPLE 4 Sensitizers Displace Activators from Antiapoptotic Proteins

Since sensitizer BH3 peptides cannot induce cytochrome c release ontheir own, but can induce cytochrome c release when activator andantiapoptotic proteins are present, in a pattern that mirrors theirbinding to antiapoptotic proteins, it was hypothesized that thesensitizers are displacing activators from the antiapoptotic proteins.As one test of this hypothesis, the ability of sensitizer peptides todisplace tBID from antiapoptotic protein Bcl-w was examined utilizing aGST-pulldown assay. Proteins bound to glutathioneagarose beads wereeluted with glutathione and analyzed by Western blot. In FIG. 3A, tBIDis displaced from BCl-w by sensitizer BH3 peptides in a pattern thatreplicates the pattern in FIG. 1D. As an additional test, thedisplacement of the activator BIM BH3 peptide from BCL-2 and MCL-1 byBAD and NOXA BH3 peptides was determined. In FIG. 3B, consistent withTable 2B, BAD BH3 efficiently displaces BIM BH3 from BCL-2, but notMCL-1, whereas NOXA A BH3 7 efficiently displaces BIM from MCL-1, butnot BCL-2. These experiments support the ability of sensitizer BH3peptides to displace activators from the antiapoptotic binding cleft.

EXAMPLE 5 A Cellular Requirement for BCL-2 Corresponds to a “BCL-2Pattern” of Mitochondrial Sensitivity to the Sensitizer BH3 Panel

In order to test whether mitochondrial dependence on individualantiapoptotic protein function can be correlated with cellular behavior,cellular models of defined antiapoptotic dependence were investigated.First, it was determined if cellular requirement for BCL-2 for cellularsurvival correlates with the BCL-2 signature of mitochondrialsensitivity to sensitizer BH3 domains found in FIG. 1B. Thepro-lymphocytic murine FL5.12 cell line requires IL-3 to maintainsurvival. Apoptosis induced by IL-3 withdrawal is inhibited byoverexpression of BCL-2 (FIG. 4A). Therefore, BCL-2-overexpressingFL5.12(FL5.12-BCL-2) cells deprived of IL-3 are a model of BCL-2dependent survival. FL5.12-BCL-2 cells grown in the presence of IL-3 areexamples of BCL-2 independent cells.

While the dependence on BCL-2 of IL-3 deprived FL5.12 cells isdemonstrated genetically in FIG. 4A, the dependence was confirmed usinga cell-permeable BCL-2 antagonist. ABT-737 has been shown to antagonizeBCL-2 (and BCL-XL and BCL-w) (Oltersdorf et al., 2005). In agreementwith the prior report, ABT-737 induced cell death in the IL-3 starved,but not the IL-3 replete BCL-2 protected cells (FIG. 4B). Moreover,ABT-737 was non-toxic to the unstressed IL-3 replete wt FL5.12 cells.This cell death was caspase dependent, demonstrating that death occurredusing the apoptotic pathway (FIG. 4C). IL-3-cells were grown in theabsence of IL-3 for 24 hours prior to initiation of treatment withcompound. All cells were treated with compounds for 24 hours prior toharvest.

Having credentialed a BCL-2 dependent cellular system, it was nextdetermined if BCL-2 dependence could be isolated at the level ofmitochondria. It was hypothesized that removal of IL-3 would “load” theBCL-2 on the mitochondria with activator BH3 proteins. It was furtherhypothesized that mitochondria bearing “loaded” BCL-2 would releasecytochrome c when treated with sensitizer BH3 peptides which compete forthe BCL-2 binding cleft. ABT-737 inhibition of BCL-2 in FL5.12-BCL-2cells primed by IL-3 withdrawal induced an apopiosis that is caspasedependent and very rapid (FIG. 5). The interpretation that the IL-3starved FL5.12-BCL-2 cells were “primed” for death is supported by therapidity of their death following ABT-737 treatment (FIG. 5).

Mitochondria were isolated from wt FL5.12 cells and FL5.12-BCL-2 cellsin the presence of IL-3, and from FL5.12-BCL-2 cells following 24 hoursof IL-3 deprivation. Due to advanced apoptosis, mitochondria could notbe isolated in sufficient quantities from wt FL5.12 cells after IL-3deprivation. FIG. 6A shows that while activators BID and BIM potentlyinduce cytochrome c release from mitochondria isolated from wt FL5.12cells, the remaining sensitizer peptides do not (blue bars). Thus,inhibition of antiapoptotic family members is by itself not sufficientto induce MOMP. Next, BCL-2 overexpression inhibits release induced by10 μM BID BH3, but not 10 μM BIM BH3, in accordance with dose-responsecurves previously demonstrated (red bars) (Letai et al., 2002). Whenmitochondria from FL5.12-BCL-2 cells deprived of IL-3 were tested,however, certain sensitizer peptides demonstrated the ability to inducecytochrome c release (tan bars), and sensitivity to 10 μM BID BH3 isrestored. It is most notable that only those sensitizer peptides withhigh affinity for BCL-2 cause MOMP. BIK BH3 does not induce cytochrome crelease in this setting, but it should be noted that it hasapproximately 10-fold lower affinity than BAD, PUMA or BMF BH3 forBCL-2. It can be seen, therefore, that cellular BCL-2 dependence can be“diagnosed” from the pattern of mitochondrial sensitivity to the panelof sensitizer BH3 peptides. This dependence can be “diagnosed” whetherthe activator involved is a recombinant protein, as in FIG. 1, or a morecomplex mix involving more than one molecule, as is likely the casefollowing IL-3 withdrawal. Note that inhibition of BCL-2 alone is notsufficient to induce cytochrome c release, as seen by the failure of allof the sensitizer peptides to induce release in the IL-3 repleteFL5.12-BCL-2 mitochondria (FIG. 6A). In fact, even the combination ofpeptides, BAD and NOXA BH3, which provide a broad spectrum ofantiapoptotic protein binding, cannot induce cytochrome c release in theabsence of an activator molecule (FIG. 6B). To induce MOMP, the BCL-2must first be “primed” by molecules communicating a death signal,generated by IL-3 withdrawal. Mitochondria isolated from FL5.12-BCL-2cells grown in the absence of IL-3 for 24 hours were treated with NOXA Aor BAD peptides (30 uM) or ABT-737 or control enantiomer at 10 uM for 35minutes. Mitochondrial pellets were subjected to chemical crosslinkingas previously described (Letai et al., 2002). BCL-2 blocks apoptosisupstream of BAX oligomerization, and BAD BH3 and ABT-737 inhibition ofBCL-2 on IL-3 starved mitochondria results in BAX oligomerization (FIG.6C). Therefore, it was hypothesized that this death signal might be anactivator BH3 protein.

BIM has previously been shown to play a role in death following IL-3withdrawal in FL5.12 cells (Harada et al., 2004). FIG. 6D shows thattotal cellular BIM levels, as well as levels of BIM complexed to BCL-2,dramatically increase following IL-3 withdrawal. It is notable thatlevels of BCL-2, BAX, and BAK stay roughly constant during the same timeperiod. These results suggest that the activator BIM (and perhaps PUMA)is a dynamic mediator of the death response following IL-3 withdrawal inFL5.12 cells, and that it is sequestered to prevent apoptosis. Cells andmitochondria bearing “loaded” BCL-2 are then “addicted” to BCL-2, anddie when BCL-2 function is antagonized. Furthermore, cellular BCL-2addiction can be diagnosed by the pattern of mitochondrial sensitivityto sensitizer BH3 domains. The negative control of immunoprecipitationusing anti-human BCL-2 antibody on lysates from IL-3 starvedFL5.12-BCL-XL cells yielded no bands, not shown.

This model predicts that BCL-2 acts upstream of BAX activation byintercepting activator BH3 molecules. To test this prediction, in FIG.6E, immunoprecipitation was performed with an antibody that recognizesonly the activated form of BAX which exposes an N-terminus epitope(Desagher ct al., 1999; Hsu and You le, 1997). wt or BCL-2 expressingFL5.12 cells were exposed to IL-3 withdrawal as indicated. IL-3withdrawal induced BAX activation in wt FL5.12 cells, while total BAXlevels remained constant. However, when BCL-2 protected against deathfrom IL-3 withdrawal, it also prevented BAX conformational change,consistent with BCL-2′s sequestering activators like BIM prior to theirinteraction with BAX (compare fourth and eight lanes). Furthermore,treatment with ABT-737 restored cytochrome c release and BAX activation,consistent with ABT-737 functioning by displacing activators from BCL-2.Taken together, these results indicate that BCL-2 blocks apoptosisupstream of BAX activation.

EXAMPLE 6 BH3 Profiling can Discriminate MCL-1 Cellular Dependence fromBCL-2 Cellular Dependence

To test if the model of antiapoptotic “priming” could be extended beyondBCL-2 to other antiapoptotic proteins, the behavior of cells protectedby BCL-2 was compared to those protected by MCL-1. 2B4 cells transfectedwith Flag-MCL-1, BCL-2, or empty vector constructs were cultured for 24hours in the presence of the indicated concentration of dexamethasone.The murine hybridoma 2B4 cell line is sensitive to dexamethasonetreatment. Overexpression of FLAGtagged MCL-1 or BCL-2 confersresistance to dexamethasone-induced apoptosis (FIG. 7A). Therefore,dexamethasone-treated, FLAG-MCL-1 expressing cells are a model ofcellular MCL-1 dependence, while dexamethasone-treated, BCL-2 expressingcells are a model of cellular BCL-2 dependence. 2B4 cells were incubatedwith dexamethasone and either ABT-737 or enantiomer for 24 hours.Treatment of the MCL-1-protected dexamethasone-treated cells withABT-737 has no effect, showing the cells are not dependent on BCL-2 forsurvival. In stark contrast, 2B4 cells protected fromdexamethasone-induced apoptosis by BCL-2 are very sensitive to ABT-737(FIG. 7B).

The cellular data provoke the prediction that mitochondria isolated from2B4-MCL-1 cells treated with dexamethasone would be sensitive to NOXAand insensitive to BAD BH3, the opposite of the pattern observed withIL-3-starved FL5.12-BCL-2 cells. Mitochondria were isolated fromdexamethasone-treated and untreated vector-transfected andFLAG-MCL-1-transfected 2B4 cells. Apoptosis was too advanced to permitisolation of mitochondria from dexamethasone treated vector-transfected2B4 cells. As can be seen in FIG. 7C, only mitochondria isolated fromthe MCL-1 dependent cells recapitulate an “MCL-1 pattern” of sensitivityto sensitizer BH3 peptides. As with the FL5.12 cells, since sensitizerBH3 peptides cause little cytochrome c release in untreated cells, it isclear that sensitizer BH3 peptide inhibition of MCL-1 (and otherantiapoptotic proteins that might be present) is not by itselfsufficient to induce apoptosis. An additional death signal (initiated bydexamethasone treatment in this case) is needed to “prime” MCL-1 so thatMCL-1 antagonism by sensitizers results in mitochondrialpenneabilization. To demonstrate the robustness of this strategy, BH3profiling was performed on 2B4 cells treated with dexamethasone, butthis time protected with BCL-2. Consistent with the priming model, aBCL-2 pattern is revealed (FIG. 7D). Thus, MCL-1 dependence, like BCL-2dependence, also can be “diagnosed” by mitochondrial sensitivity to thesensitizer BH3 panel.

As with the FL5.12cells, it was determined if dexamethasone treatmentresulted in increased sequestration of an activator BH3 protein by MCL-1and BCL-2. Vector or FLAG-MCL-1 transfected 2B4 cells were treated with0 or 100 nM dexamethasone and lysed. FIG. 7E shows that FLAG-MCL-1sequesters increased amounts of BIM following the death signalinginduced by dexamethasone treatment, as does BCL-2 (FIG. 7F). Note thatlevels of BAX and BAK stay constant during the treatment. Also note thatit appears that the small amount of BAX bound to cells before treatmentwith dexamethasone decreases after treatment. One interpretation is thatthe BAX is displaced by increased levels of BIM binding to BCL-2. Thisis significant because it suggests that displacement of BAX from MCL-1is insufficient to induce MOMP and death.

To further demonstrate that the mitochondrial assays reflect truecellular dependence, peptides were transfected via electroporation intoFLAG-MCL-1-transfected 2B4 cells that had been treated withdexamethasone, putatively priming MCL-1 with death signals, carried atleast in part by BIM. Percent killing was ascertained by Annexin-Vstaining. Note that killing by transfection with NOXA A is significantlygreater than that with BAD, recapitulating the same MCL-1 patternobserved in isolated mitochondria from this same cell line (FIG. 7G,compare with Table 2B, FIG. 1E, FIG. 7C). N=3 and the error barsrepresent the standard deviation. These results support the cellularrelevance of the mitochondrial BH3 profiling assays.

EXAMPLE 7 Dependence on BCL-2 in a Leukemia Corresponds to MitochondrialSensitivity to Sensitizers in a “BCL-2 Pattern” and Sequestration of BIM

Dependence on antiapoptotic proteins is perhaps of greatest importancein the context of cancer, in which antiapoptotic BCL-2 family proteinsare subjects of intense investigation as therapeutic targets. While theconcept of oncogene addiction has received attention recently (Jonkersand Berns, 2004; Weinstein, 2002), the molecular details of theaddiction to specific oncogenes is poorly understood. A validated modelof oncogene addiction, a BCL-2 dependent murine leukemia, was used toexamine the molecular basis for BCL-2 “addiction.”

Previous results have described a mouse acute lymphocytic leukemia modelin which c-myc is constitutively expressed and human BCL-2 isrepressibly expressed. In this model, when BCL-2 transgene expression iseliminated by administration of doxycycline, the leukemic cells undergoapoptosis, resulting in rapid resolution of the leukemia (Letai, 2004).This provides an ideal in vivo model of a BCL-2 dependent cancer. It washypothesized that dependence on BCL-2 was due to a similar mechanism tothat of the IL-3 deprived FL5.12-BCL-2 cells—that is, a death signal wasbeing initiated and carried by an activator BH3 molecule, but BCL-2 wasbinding it and preventing its interaction with multidomain proapoptoticproteins.

Mitochondria were isolated from leukemia cells and exposed to sensitizerBH3 peptides. Subsequently, cytochrome c release was measured. As aninternal control, mitochondria were isolated from liver from theleukemic mice in parallel (FIG. 8A). The sensitizer BH3 peptides wereunable to induce cytochrome c release from non-malignant hepatocytemitochondrias from the leukemic mice, just as they were unable to inducecytochrome c release from non-malignant liver mitochondria from normalmice (FIG. 1A) or from non-malignant FL5.12 (FIG. 6A) or 2B4mitochondria (FIG. 7C). Intriguingly, certain sensitizer BH3 peptideswere capable of inducing near total cytochrome c release from theleukemic mitochondria (FIG. 8B). Significantly, the pattern of peptideswhich induced release corresponded exactly to those peptides which bindwith high affinity to BCL-2 (Table 2B), namely BAD, BIK, PUMA, and BMF.Note that, consistent with its approximately ten-fold lower affinitythan BAD BH3 for BCL-2, BIK BH3 requires a ten-fold higher concentrationto demonstrate cytochrome c release. A ten-fold increase in NOXA Apeptide concentration has no effect, consistent with the extremely lowaffinity NOXA A has for BCL-2.

These results suggest that in this leukemia model, death signals arebeing continually initiated, and BCL-2 is required to sequester theactivator BH3 molecule to prevent apoptosis. In contrast to thenon-malignant systems tested above, leukemic cell BCL-2 behaves as ifalready “primed” with activator protein(s) without any furtherintervention, such as growth factor withdrawal or dexamethasonetreatment. FIG. 8C shows that BIM is expressed in the leukemia cells,and it is bound by BCL-2. Supporting the signal importance of BIM intransmitting death signals in this model, BID is also present in thelysate, but is not bound by BCL-2. Note that PUMA is also found to bebound by BCL-2, consistent with a report showing PUMA deficiency couldaccelerate myc-induced lymphomagenesis (Hemann et al., 2004). Since thePUMA BH3 lacks the ability to directly activate BAX or BAK, it washypothesized that PUMA is acting as a sensitizer in this context, ineffect decreasing the amount of BCL-2 available to bind BIM and possiblyBAX or BAK.

If BCL-2 maintains survival of this leukemia cell primarily bysequestering BIM, then one would predict that BIM loss of function couldsubstitute for BCL-2 overexpression to cooperate with c-myc inleukemogenesis. In fact, this experiment has already been performed. Itwas found that BIM deficiency can indeed cooperate with c-myc to producea pre-B lymphocytic leukemia like the one produced here by thecooperation of BCL-2 overexpression with c-myc (Egle et al., 2004).These results support a model in which BCL-2 is necessary for survivalof leukemia largely because it is required to sequester BIM, preventingactivation of BAX/BAK and subsequent MOMP. The leukemia cells aretherefore neither normal and healthy, nor dead, but rather primed fordeath.

EXAMPLE 8 BH3 Profiling Predicts Sensitivity to ABT-737

As another test of the ability of BH3 profiling to detect in vivo BCL-2dependence, two small cell lung cancer (SCLC) cell lines were examined.Mitochondria were isolated from two SCLC cell lines, H146 and H1963 andexposed to panel of BH3 peptides. Cytochrome c release quantitated byELISA. Both were sensitive to treatment with ABT-737 in vitro and in anin vivo murine xenograft model (Oltersdorf T, 2005). Both H146 and H1963demonstrate a pattern of sensitivity diagnostic of BCL-2 sensitivity(FIG. 8D). This provides further support, in addition to the results ofFIG. 4B and FIG. 7E, that mitochondrial BH3 profiling is a powerfulpredictor of what cells are sensitive to BH3 mimetic drugs in vitro andin vivo.

EXAMPLE 9 ABT-737 Kills CLL Cells in the Low Nanomolar Range

ABT-737 is a cell-permeant small molecule with affinity to BCL-2,BCL-XL, and BCL-w in the sub-nanomolar range. The negative controlenantiomer (enant) is a stereoisomer of ABT-737 that binds to BCL-2 withlower affinity, and has been used as a loss of function control(Oltersdorf et al., 2005). CLL cells have shown sensitivity to ABT-737,so they were selected as a possible initial cancer model of BCL-2dependence (Oltersdorf et al., 2005). Freshly isolated primary CLL cellswere incubated with ABT-737 or negative control enantiomer. After 48hours, death was assessed using Annexin-V staining. In all twenty-fourCLL samples tested, apoptosis was induced within 48 hours by ABT-737with an EC50 of 4.5+2.2 nM (FIG. 12A) (range 1.9-9.4 nM, see Table 3).The negative control enantiomer was less potent (mean EC50 574 nM) (FIG.12A). Directly targeting the apoptotic machinery might be expected toinduce apoptosis rapidly. Five samples treated for four hours respondedsimilarly compared to 48-hour treatment (FIG. 12B). Non-malignantperipheral blood mononuclear cells (PBMC) from normal donors wereresistant to ABT-737 with an EC50 >1000 nM (FIG. 12C).

TABLE 3 EC50 ABT- EC50 WBC LDH 737 NCE RaI (×10E- (313- beta-2 treatmentIgVH Patient (nM) (nM) Age Stage 3/ul CD38 618) (0-2.7) History StatusV1 7.0 1114 74 2 282 nd 819 4.1 untreated nd V2 3.2 473 75 1 72.5 nd 3373.7 untreated mut V3 3.5 622 63 3 89.4 neg 2138 2.5 untreated un V4 3.6496 57 1 164.6 nd 563 5.6 C; R mut V5 2.1 619 62 2 61.2 nd 431 5.3untreated mut V6 3.1 493 65 2 126 neg 533 4.5 F; F + Cy; R mut V7 1.91783 58 2 47.1 nd 444 1.7 untreated nd V8 4.1 500 43 2 216.4 nd 398 3untreated mut V9 8.7 1009 76 1 108.3 nd 434 2.6 untreated un V10 5.8 63055 1 66.3 pos 613 3.2 untreated un V11 3.1 334 57 1 85.3 nd 501 1.9untreated nd V12 6.0 679 74 2 257.1 nd 473 3.2 untreated mut V13 4.5 68341 1 307.9 pos 1310 5.3 untreated un V14 3.3 318 89 2 233.9 neg 463 1.8untreated mut V15 6.4 582 63 3 201.8 nd 431 3.2 untreated un V16 2.9 34360 1 72.2 nd 444 1.9 untreated mut V17 9.4 861 47 3 244.1 nd 424 2.6untreated mut V18 2.3 317 46 1 89.9 nd 450 2 untreated mut V19 6.4 57255 1 162 nd 156 2.7 untreated mut V20 4.8 119 66 3 502 neg 563 5.3S:C:F:R mut V21 2.1 215 68 1 107.9 nd 647 3.5 untreated mut V22 4.0 31062 3 343 nd 583 4.3 untreated mut V23 7.4 239 64 7 140.5 nd 458 2.4untreated mut V24 6.1 616 55 1 55 pos 503 2.6 untreated mut nd = notdetermined C = chlorambucil R = rituximab F = fludarabine Cy =cyclophosphamide S = splenectomy

EXAMPLE 10 BCL-2 and BIM Levels are Consistent among CLL Samples

Given the consistency of response to ABT-737, it was hypothesized thatBCL-2 protein expression was also uniform in CLL cells. In addition,proapoptotic BIM has been shown to be an important determinant ofcommitment to apoptosis in lymphocytic cells (Bouillet et al., 1999;Opfennan et al., 2003). Antiapoptotic BCL-2 and proapoptotic BIM levelsamong 15 CLL samples were remarkably uniform (FIG. 13A); levels of BCL-2and BIM in PBMC's were consistently lower (FIG. 13B). To ensure thatshort-term culture did not affect BCL-2 or BIM levels and perhaps alterresponse to ABT-737, protein lysates were made from CLL samples at timeof isolation and after 48 hours in culture. Neither BCL-2 nor BIM levelschanged during culture (FIG. 13C). BCL-2 levels in CLL cells werecompared to levels in follicular lymphoma, a cancer in which BCL-2 isoverexpressed due to the t(14;18) translocation (FIG. 13D). BCL-2 levelswere notably similar in the two diseases.

Table 4 summarizes the clinical characteristics of the source patientsin FIG. 12A. EC50 values were compared between groups dichotomized byfactors previously identified as prognostically useful in CLL. Thisanalysis revealed that in no case did a difference in mean EC50 betweengroups exceed 2 nM. A nonparametric statistical comparison of the groupsshowed that none differed with statistical significance except thegroups dichotomized by leukocyte count (Table 5). Thus, biologicalresponse to ABT-737 appears to be largely independent of traditionalprognostic factors.

TABLE 4 # Median Range Rai stage Age 82.5 41-70 0 0 years 1-2 19Leukocyte count 133.2  47-502 3-4 5 at range 4.8-10.8 × CD28 1000/ulpositive 3 LDH 487  185-2138 negative 4 at range 813-648 ul not assessed17 S2 microglobulin 3.1 1.7-5.5 at range 0-2.7 mg/ml Treatment historyprior treatment 5 untreated 21 IgvH status mutated 13 immolated 5 notassessed 3 ZAP79 positive 9 negative 9 not assessed 9

TABLE 5 parameter dichotomy p value CD38 pos vs neg 0.11 beta-2 0-27vs >2.7 0.56 age 0-55 vs >65 0.14 LDH 0-618 vs >618 0.89 WBC 0-133vs >133 0.02 Higher WBC correlates with higher EC50 Rai stage 0-2 vs 3-40.14 prior treatment yes vs no 0.93 IgVH mutated pos vs neg 0.10 ZAP70pos vs neg 0.69

EXAMPLE 11 CLL Mitochondria Reveal a Tonic Dependence on BCL-2 Functionto Maintain Outer Membrane Integrity

Since BCL-2 opposes the intrinsic, or mitochondrial, pathway ofapoptosis, it was hypothesized that the toxicity of ABT-737 was based ona mitochondrial requirement for BCL-2 function in CLL. A panel ofpeptides has been characterized, which derived from the BH3 domains ofBH3-only proteins that behave as selective antagonists of BCL-2 functionin several genetically defined model systems (Letai et al., 2002) (and MC, V D M, Nishino M, Wei G, Korsmeyer S, Annstrong S, A L, inpreparation). For instance, BH3 peptides from BAD, PUMA, BMF, and, withlower potency, BIK bind and inhibit BCL-2 function, whereas BH3 domainsfrom NOXA, HRK and BNIP-3A do not interact with BCL-2. This panel hasbeen validated on other antiapoptotic family members, and this patternof interaction is distinct for each antiapoptotic protein, so that thefunction of each may be specifically detected by this “BH3 profiling.”Therefore, mitochondria that depend on BCL-2 function for maintenance oftheir outer membrane integrity show induction of outer membranepermeability when treated with BAD, PUMA and BMF, but not NOXA, HRK, andBNIP-3A peptides.

CLL mitochondria were incubated with BH3 peptides as well as ABT-737 andnegative control enantiomer (FIG. 14). Note that activators BID and BIMBH3 peptides interact with all antiapoptotic proteins tested andfurthermore can directly activate BAX and BAK (Letai, et al., 2002), sothat they act as positive controls for cytochrome c release assays. BAD,PUMA and BMF induce cytochrome c release, whereas the NOXA, HRK, andBNIP-3A peptides, and a point null mutant of BAD BH3, do not. Thispattern is diagnostic of mitochondrial BCL-2 dependence. ABT-737 alsoinduced cytochrome c release, validating that its target is located atthe mitochondria of CLL cells and required to maintain mitochondrialouter membrane integrity. Therefore, these “BH3 profiling” experimentsdemonstrate that the CLL mitochondria depend on BCL-2 function tomaintain outer mitochondrial membrane integrity, elucidating a mechanismfor the exquisite sensitivity of CLL cells to ABT-737 treatment. Sincethe BH3 peptides in the sensitizer panel lack the ability to directlyactivate BAX and BAK, these experiments also implicated the presence ofan activator molecule constitutively sequestered by BCL-2 in CLL.

EXAMPLE 12 BIM Bound to BCL-2 Primes CLL Cells For Killing By ABT-737

Previously studies have shown that ABT-737 and the sensitizer BH3peptides act as antagonists of antiapoptotic BCL-2, but lack the abilityto directly activate BAX and BAK. In order to induce MOMP, sensitizersrequire the presence of an activator like BIM or BID (Letai et al.,2002; Oltersdorf et al., 2005). The results above, therefore, suggest anactivator is bound to BCL-2, then displaced by ABT-737 or the BCL-2binding BH3 peptides. Following displacement, it was hypothesized thatthe freed activator could induce MOMP via interaction with BAX and BAK.

FIG. 13 demonstrated the presence of BIM in CLL samples. Levels of BID,the other established activator BH3-only protein, were very faint byimmunoblot, and cleaved, activated BID was almost to completelyundetectable (not shown). It was hypothesized that BIM was indeed boundby BCL-2. FIG. 15A shows that BIM is sequestered by BCL-2 in primary CLLcells. Furthermore, treatment with ABT-737, and to a lesser degree, theless potent negative control enantiomer, causes a dramatic reduction inthe amount of BIM bound to BCL-2 (FIG. 15B). Interestingly, displacedBIM seems to be rendered less stable, as total cellular levels of BIM,but not BCL-2, are reduced following ABT-737 treatment. Co-treatmentwith a pan-caspase inhibitor ZVAD-fmk protects against ABT-737-induceddeath, further implicating an apoptotic death. ZVAD-fmk also preservescellular BIM levels, suggesting that displaced BIM may be cleaved bycaspases as previously reported (Chen and Zhou, 2004). Detergents inlysates can interfere with the binding of sensitizers to the BCL-2hydrophobic cleft (not shown), so it was hypothesized that the observedpersistence of BIM bound to BCL-2 after ABT-737 and ZVAD-fmk treatmentmight be an artifact due to detergent in the lysate inhibiting ABT-737'sbinding to BCL-2. Two competing hypotheses of ABT-737 mechanism ofaction were generated. In the first, ABT-737 displaces BIM from BCL-2,inducing BAX oligomerization and caspase activation, and finally caspasecleavage of the displaced BIM. In the second, BIM degradation is merelya consequence of MOMP and caspase activation that is initiated by amechanism independent of BIM displacement.

To test these competing hypotheses, whole cell lysates were examined forBCL-2:BIM complex levels in the presence or absence of ABT-737. Indetergent-free conditions, ABT-737, but not the negative controlenantiomer, displaces BIM from BCL-2 into the supernatant (FIG. 15C).BIM has been shown to activate BAX and induce its oligomerization (Letaiet al., 2002; Marani, et al., 2002; Yamaguchi and Wang, 2002).Consistent with this mechanism, CLL cells display oligomerization of BAXwithin four hours of treatment with ABT-737 (FIG. 15D).

If the first hypothesis is correct, and BIM is required for inducing theMOMP following BCL-2 antagonism, selective sequestration of BIM shouldcause a reduction in cytochrome c release following antagonism of BCL-2on CLL mitochondria. In FIG. 15E, BCL-2 function is antagonized with thesensitizer BAD BH3 peptide. As shown in FIG. 12, BAD BH3 by itselfinduced cytochrome c release. However, addition of an antibody thatbinds the BH3 domain of BIM caused a dramatic reduction in cytochrome crelease. An irrelevant antibody had no effect. Prevention of cytochromec release by masking BIM supports the first hypothesis, that antagonismof ABT-737 is toxic to CLL cells due to the displacement of BIM (FIG.15C) from a BCL-2:BIM complex. Displaced BIM then induces BAXoligomerization (FIG. 15D), MOMP and commitment to programmed celldeath. An important implication of these results is that BCL-2expression is necessary but not sufficient to dictate response toantagonism of BCL-2 by ABT-737 or sensitizer BH3 peptides. ActivatorBH3-only proteins like BIM must be sequestered by BCL-2 for the cell tobe sensitive to BCL-2 antagonism.

EXAMPLE 14 Predication of Drug Response by BH3 Profiling

BH3 profiling allows for the prediction of response of cancer cells toanti-cancer therapeutics. For the purposes of this application,therapeutics can be divided into those that target anti-apoptotic BCL-2proteins, and conventional agents. As a model test system, four lymphomacell lines SU-DHL4, SU-DHL6, SU-DHL8, and SU-DHL 10 were employed. Toperform BH3 profiling, the ability of a panel of sensitizer peptides toinduce mitochondrial outer membrane permeabilization (MOMP) inmitochondria isolated from the lymphoma cells were tested. For easyreference, FIG. 18A shows the interaction pattern between the BH3peptides and anti-apoptotic proteins. MOMP was measured by quantifyingcytochrome c release by ELISA.

As shown in FIG. 18B-E, BH3 profiling proved able to distinguish thesethree classes of blocks in our sample of four lymphoma lines. SU-DHL4and SU-DHL6 a “primed” phenotype, based on the sensitivity to sensitizerBH3 peptides. Note that a strong response to the PUMA BH3 peptide, whichinteracts with all of the antiapoptotic proteins, provides a usefulgauge of whether the mitochondria are primed. The pattern of sensitivity(PUMA, BMF, BAD, +/−BIK) indicated a dependence on BCL-2 for SU-DHL-4.SU-DHL 6 also was primed, as shown by a strong PUMA BH3 and BMF BH3signal. The weaker, but definite, response to both of the more selectiveBH3 peptides BAD BH3 and NOXA A BH3 implicate combined dependence onBCL-2 and MCL-1. SU-DHL-8 appeared to be poorly primed, given thelimited response to PUMA BH3 and other sensitizers, but nonethelessdemonstrated an intact effector arm by responding strongly to activatorsBIM BH3 and BID BH3. SU-DHL-10 responded poorly to both sensitizer andactivator peptides, indicating the loss of the effector arm.

Only cells that are dependent on BCL-2 for survival are predicted torespond to BCL-2 antagonists like ABT-737. Therefore, BH3 profilingpredicts that SU-DHL4 and SU-DHL6 should respond to ABT-737. Thishypothesis was tested and confirmed that BH3 profiling accuratelypredicted response to ABT-737 (FIG. 19A). Furthermore, BH3 profilingwould predict that SU-DHL4 and SU-DHL6 might be more sensitive to otherchemotherapy agents, as they are “primed” for death and SU-DHL-8 andSU-DHL-10 were not. To test this, cells were treated with vincristine.As predicted by BH3 profiling SU-DHL4 and SU-DHL6 were the mostsensitive cell lines (FIG. 19B).

EXAMPLE 15 Cell Based BH3 Profiling

A method that converts the mitochondrial-based BH3 profiling to acell-based assay was developed. In this assay, cells are permeabilizedby digitonin to permit peptide access to mitochondria. Cells are treatedwith the fluorescent dye JC-1 to evaluate loss of mitochondrialtransmembrane electrochemical potential due to treatment with thepeptides. Loss of mitochondrial integrity due to apoptosis can beobserved by a shift in the fluorescence peak from 590 nm to 520 nm.Multiple assays may be read in parallel on a 96- or 384-well plate inthe TECAN Safire2 fluorimeter. To test this system, it has been appliedit to several cell lines of known BCL-2 dependence, and have obtainedexcellent correlation between results of mitochondrial BH3 profiling andcellular BH3 profiling. Two examples may be seen in FIGS. 20A-20D.

Other Embodiments

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

1. A method of predicting sensitivity of a cancer cell to a therapeuticagent comprising contacting said cancer cell with a BH3 domain peptideand detecting apoptosis of said cancer cell, wherein the presence ofapoptosis indicate said cancer cell is sensitive to a therapeutic agent.2. A method of predicting sensitivity of a cancer cell to a therapeuticagent comprising contacting mitochondria from said cancer cell with aBH3 domain peptide or mimetic thereof and detecting cytochrome C releasefrom said mitochondria, wherein the cytochrome C release indicates saidcancer cell is sensitive to a therapeutic agent.
 3. A method ofselecting an agent that is therapeutic for a subject comprising: a)providing a cancer cell from said subject; b) contacting said cancercell with a BH3 domain peptide or mimetic thereof; c) determiningwhether or not said BH3 domain peptide or mimetic thereof inducesapoptosis in said cancer cell to produce a test BH3 profile; d)comparing said test BH3 profile with a therapeutic agent BH3 profile;wherein a similarity of said test BH3 profile compared to saidtherapeutic agent BH3 profile indicates that the agent is therapeuticfor said subject.
 4. A method of predicting sensitivity of a cancer cellto a therapeutic agent comprising providing a BH3 profile of said cancercell and comparing said BH3 profile to a control profile, wherein asimilarity of said BH3 profile of said cancer cell compared to saidcontrol profile indicate said cancer cell is sensitive to saidtherapeutic agent.
 5. The method of claim 1, wherein said cancer cell ispermeabilized prior to contacting with said BH3 domain peptide.
 6. Themethod of claim 5, further comprising contacting said permeabilized cellwith a potentiometric dye.
 7. The method of claim 6, where saidpotentiometric dye is JC-1 or dihydrorhodamine
 123. 8. The method ofclaim 6, where said apoptosis is measured by detecting a change inemission of said potentiometric dye.
 9. The method of claim 1, whereinsaid BH3 domain peptide is derived from the BH3 domain of a BID, a BIM,a BAD, a BIK, a NOXA, a PUMA a BMF, or a HRK polypeptide.
 10. The methodof claim 1, wherein said BH3 domain peptide is selected from the groupconsisting of SEQ ID NO: 1-14 and
 15. 11. The method of claim 1, whereinsaid therapeutic agent is a chemotherapeutic agent, BH3 domain mimetic,or antagonist of an anti-apoptotic protein.
 12. A profile, comprising apattern of mitochondrial sensitivity to BH3 domain peptides selectedfrom the group consisting of SEQ ID NO: 1-12 and 13 taken from one ormore subjects who have cancer.
 13. A cell based assay system comprisinga permeabilized labeled cell and a BH3 domain peptide or mimeticthereof.
 14. The assay system claim 13, wherein said labeled cellcomprises a potentiometric dye.
 15. The method of claim 14, wherein saidpotentiometric dye is JC-1 or dihydrorhodamine
 123. 16. The method ofclaim 3, wherein said cell is permeabilized prior to contacting withsaid BH3 domain peptide or mimetic thereof.